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Antitoxins. 


O;  e.  KRIEGER. 


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Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrpsoft  Corporation 


http://www.archive.org/details/bloodserumtherapOOkrierich 


Blood  Serum  Therapy 
AND  Antitoxins. 


BY 

GEORG   E.  KRIEGER,  M.  D., 

Surgeon  to  the  Chicago  Hospital,  etc. 


WITH  ILLUSTRATIONS. 


Chicago  : 

E.  H.  COLEGROVE  &  CO 

1895. 


Entered  according  to  Act  of  Congress,  in  the  year  1895, 

By  GEORG  E.  KRIEGER,  M.  D., 

in  the  office  of  the  Librarian  of  Congress,  at  Washington,  D.  C 


k^  P  ^ 


Uvbvav 


TO  THE  MEMORY 

OF 

MY  BELOVED   FATHER,  THE  LATE 

GEHEIMER  MEDICINALRATH  DR.   E.KRIEGER, 

MEMBER  OF 

THE  MEDICINAL  COLLEGIUM,  ETC.,  ETC., 

AT   BERLIN. 


m350:273 


CONTENTS. 


PAGE. 

Preface 5,  6 

Chapter  I. — Blood  Serum  Therapy 7 — 15 

Chapter  II  — Toxins  and  Toxalbumins 16 — 24 

Chapter  III.— Tetanus 25—41 

Chapter  IV.— Diphtheria 42—69 


PREFACE. 


It  has  always  been  the  effort  of  human  intelligence 
to  find  remedies  of  specific  value  in  certain  diseases  and 
the  belief  to  have  discovered  a  valuable  drug  has  brought 
upon  the  market  a  legion  of  chemical  preparations.  Pre- 
pared by  the  chemist,  examined  by  the  physiologist  as  to 
their  effect  upon  the  organism  they  found  their  way  into 
the  hands  of  a  clinical  observer  who  would  soon  startle 
the  world  by  the  exhibition  of  the  new  "panacea''  he 
discovered.  The  drug  was  quickly  tried  in  every  hospital, 
and  on  the  strength  of  medical  testimonials  and  mercan- 
tile enterprise  it  became  in  a  short  time  a  much  demanded 
^'infallible  remedy."  However  as  suddenly  as  it  had 
gained  fame,  it  sunk  again  into  oblivion.  This  empirical 
wa}'^  of  detecting  specific  remedies  was  well  commented 
on  by  the  following  utterance  of  Rossbach  :  '*  Since 
thousands  of  years  mankind  has  experimented  in  this  di- 
rection and  the  result  has  been  the  discovery  of  but  four 
remedies  for  three  diseases.  It  would  be  a  terrible  idea 
that  some  other  thousand  years  were  necessary  to  detect 
another  four  remedies.  The  usual  way  of  proceeding  is 
too  dangerous.  Especially  harmful  proves  the  enthusiasm 
not  to  say  dishonesty  of  many  observers.  If  by  chance 
a  physician  has  found  a  remedy,  after  the  application  of 
which  one  or  two  cases  of  an  infectious  disease  have 
quickly  recovered,  the  success  is  at  once  attributed  to  the 
medicine.  No  thorough  investigation  on  a  larger  scale  is 
made,  the  new  "specific  "  has  been  discovered  and  is  em- 
phatically recommended. 


The  result  of  all  this  enthusiastic  praise  is  that  the 
profession  has  lost  confidence  and  can  hardly  be  convinced 
even  if  more  valuable  proofs  are  offered." 

At  present  we  find  ourselves  in  a  different  situation. 
We  try  to  explain  the  etiology  of  a  disease,  we  endeavor 
to  ascertain  the  noxious  principles  and  finally  to  prepare 
such  substances  as  would  act  in  the  opposite  direction, 
and  so  protect 'the  organism  against  infection. 

The  experiment,  however,  is  not  made  on  men  before 
the  value  of  the  method  has  been  fully  established  on  ani- 
mals. Thus  having  found  a  preparation  which  possesses 
specific  curative  properties  in  human  diseases,  transferred 
to  animals,  we  try  to  precisely  determine  the  safety  of  its 
application  and  the  limits  of  its  therapeutic  value.  When 
after  these  preliminary  studies  the  remedy  is  employed  in 
the  practice  of  medicine  we  need  no  more  investigations  as 
to  its  specific  effect,  we  know  its  characteristic  influence 
and  only  have  to  corroborate  in  men  the  result  of  former 
experiments.  The  only  question  still  open  for  discussion 
is  the  exact  determination  of  the  dosage  in  the  various 
forms  of  the  disease  and  the  different  condition  of  the  pa- 
tients. The  above  course  has  been  followed  in  establish- 
ing the  virtues  of  serum  therapy  and  antitoxins,  the 
chemical,  bacteriological  and  therapeutic  effect  of  which 
will  be  the  subject  of  the  following  essay. 


CHAPTER  I. 


Blood  Serum  Therapy. 

In  order  to  intelligently  understand  what  is  meant  by 
blood  serum  therapy  and  to  comprehend  its  revolutionizing 
importance  in  medical  science  we  have  to  look  back  for  its 
origin,  and  for  those  facts  which  are  fundamental  for  this 
modern  method  of  treating  infectious  diseases.  The  prin- 
ciple of  serum  therapy  is  based  upon  the  discoveries  of 
Prof.  Behring,  of  Berlin,  that  the  blood  serum  of  an  ani- 
mal organism,  which  has  been  rendered  immune  against  a 
certain  infectious  disease,  has  the  power  when  injected 
into  another  living  organism,  to  protect  the  latter  against, 
or  even  to  cure  it  after  infection  of  this  disease.  For  in- 
stance, a  guinea  pig  or  mouse,  which  are  very  suscepti-  Y 
ble  to  tetanus  poison,  can  almost  with  certainty  be  saved 
after  inoculation  with  said  poison  or  the  virulent  tetanus 
germs  if  a  sufficient  quantity  of  serum  of  a  horse  pre- 
viously rendered  immune  against  tetanus  is  injected  into 
them. 

The  practical  application  of  this  theory  in  medicine 
anticipates  a  thorough  knowledge  of  the  principles  of  the 
doctrine  of  immunity  and  I,  therefore,  will  consider  these 
in  particular  before  speaking  of  serum  therapy  in  general. 

It  is  commonly  known  that  one  class  of  animals  is 
more  susceptible  to  a  certain  disease  than  another  and 
that  some  are  by  nature  protected  or  naturally  immune 
against  the  same  disease.  Among  those  classes  that  are 
susceptible  for  instance  to  diphtheria,  may  be  some  in- 
dividuals which  possess  a  personal  immunity  against   th  e 


8  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

infection.  Such  personal  or  individual  immunity  is  either 
natural  or  may  be  acquired.  The  acquired  immunity  is 
effected  by  accidental  or  by  artificial  infection,  the  former 
in  case  of  a  previous  sickness,  the  latter  by  protective 
inoculation.  A  child  who  has  recovered  from  scarlet 
fever  is  usually  thereafter  immune.  An  animal  inoculated 
with  anthrax  poison  is  after  recovery,  rendered  artificially 
immune. 

Old  as  these  principles  are,  one  hundred  years  hav- 
ing elapsed  since  they  were  first  applied  in  medicine  by 
employing  vaccination  against  smallpox  (Jenner),  they 
were  not  understood  scientifically  until  Pasteur  made  his 
eminent  discoveries  concerning  the  protective  inoculation 
of  animals,  and  by  scientific  study  of  the  facts  thus  ob- 
tained gave  the  impulse  for  the  development  and  knowl- 
edge of  a  new  and  highly  important  science.  In  1880, 
he  found  that  the  culture  of  bacilli  of  chicken-cholera 
when  dried  and  exposed  to  the  air  for  several  months, 
lost  some  of  their  virulence,  and  have  not  that  fatal  effect 
on  chickens  as  when  fresh  and  fully  virulent,  and  further- 
more that  chickens  inoculated  with  such  attenuated  culture 
were  made  immune  against  the  virulent  cultures. 

At  the  time  of  these  discoveries  it  was  hardly  com- 
prehended by  the  profession  how  important  a  fact  had 
thereby  been  brought  to  our  knowledge,  but  it  was  soon 
recognized  when  the  method  of  protective  inoculation 
showed  equally  good  results  in  anthrax,  erysipelas  of  hogs 
and  other  infective  diseases  among  animals. 
:ion  of  Xhe  principal  feature  in  obtaining  the  proper  material 

^  for  inoculation  is  the  attenuation  of  the  pathogen  cultures. 
Among  the  various  ways  of  proceeding,  the  first  was  their 
desiccation  by  simple  exposure  to  the  air,  the  oxygen  of 
which  seems  to  be  instrumental  in  their  attenuation.  Soon, 
however,    the   latter   was    more    practically   accomplished 


BLOOD  SERUM  THERAPY.  9 

by  adding  chemical  substances  to  the  culture,  such  as 
weak  solution  of  sulphuric  acid,  peroxide  of  hydrogen, 
carbolic  acid,  bichloride  of  mercury,  etc.  Further  experi- 
ments led  to  another  method  with  as  good  effect  :  The 
attenuation  by  thermic  influence.  According  to  Toussaint 
(1880),  the  anthrax  culture  loses  its  virulence  when  kept  at 
fifty-five  degrees  Cels.  for  ten  minutes. 

It  took,  of  course,  a  long  line  of  experiments  to  ascer- 
tain at  what  degree  the  various  microbes  should  be  kept  in 
order  to  render  them  most  useful  for  protective  inoculation, 
and  regarding  some  of  them  it  has  been  found  that  they 
are  best  prepared  for  the  purpose  by  keeping  them  at  a 
temperature  just  below  the  degree  which  would  entirely 
destroy  their  vitality.  Still  another  method  was  obtained 
by  Pasteur's  discovery  that  the  microbes  of  a  disease  which 
exclusively  belong  to  a  certain  class  of  animals  lose  their 
virulence  when  transplanted  to  a  different  species,  as  for 
instance  those  of  hog  erysipelas  to  rabbits,  those  of 
chicken-cholera  to  hogs,  etc.  By  all  these  different 
methods  the  living  microbes  are  by  no  means  killed,  but 
their  vitality  is  weakened  and  their  attenuation  is  accom- 
plished by  changing  the  naturally  favorable  condition  for 
their  development  and  multiplication  for  conditions  unfa- 
orable  to  their  growth. 

The  question  in  which  way  the  inoculation  affects  the  immunization. 
organism  and  how  the  latter  becomes  immune  has  been 
subject  to  various  theories.  At  first  Pasteur  and  others 
were  of  the  opinion  that  the  microbes  after  having  been 
multiplied,  consume  the  necessary  nourishing  substances 
so  that  a  lack  of  food  is  caused  for  those  that  afterward 
may  enter  the  system.  Another  theory  and  more  apt  to 
be  correct  is  that  of  Chauveau,  who  believes  that  during 
the  process  of  immunization  certain  bacterial  products  are 
formed   which  are  detrimental    to  the    microbes,    thereby 


10  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

rendering  a  second  infection  impossible.  Entirely  differ- 
ent, however,  became  the  views  on  this  subject  when  Sal- 
mon and  Smith  discovered  (1887)  that  immunization  can 
be  accomplished  without  inoculating  the  living  bacteria, 
namely,  by  purely  chemical  agents.  This  discovery  is  based 
upon  the  fact  that  the  bacterial  products  separated  from  the 
microbes  had  when  inoculated  the  same  immunizing  effect  to 
the  organism  as  a  living  culture.  Thus  the  chemical  na- 
ture of  the  immunizing  agent  became  more  apparent,  and 
the  change  of  substance  in  the  organism  itself,  brought 
on  by  inoculation,  was  considered  the  real  cause  for  its  in- 
creased resistance  against  the  respective  microbes. 

Simultaneously  with  this  discovery  more  facts  pertain- 
ing to  the  question  of  protective  inoculation  were  brought 
to  light,  partly  explaining  and  partly  corroborating  the 
results  already  obtained.  It  was  observed  that  the  lymph 
and  blood  of  a  normal  healthy  organism  possesses  bacteri- 
cide properties.  This  fact  was  substantiated  by  the  ex- 
periment to  cultivate  microbes  in  the  blood  of  an  animal 
just  killed.  A  large  number  of  the  bacterial  cells  perished 
and  it  took  several  hours  before  the  bactericide  power  of 
the  fluid  decreased  to  such  an  extent  as  to  allow  the  re- 
mainder of  living  bacteria  to  be  developed  in  the  now  in- 
active fluid.  Owing  to  the  ingenious  investigations  of 
Fodor,  Nuttal,  Behring,  and  Buchner,  we  learned  that  these 
bactericide  properties  are  due  to  certain  albuminoids  which 
are  dissolved  in  the  blood  serum,  and  furthermore,  that  the 
serum  of  different  classes  of  animals  show  a  different  bac- 
tericide ability.  From  these  facts  the  conclusion  was 
drawn  that  animals  which  are  naturally  immune  against  a 
certain  disease  would  furnish  serum  with  neutralizing 
power  and  the  experiments  of  Behring  really  proved  these 
to  be  a  fact.  Behring  found  that  the  blood  and  blood 
serum  of  a  rat  which  is  naturally  immune  against  anthrax 


BLOOD  SERUM  THERAPY.  11 

has  strong  bactericide  properties,  while  that  of  rabbits, 
mice,  cattle,  etc.,  which  are  very  susceptible  for  anthrax  in- 
fection have  none. 

The  same  author  in  further  studying  this  subject  soon  Behnngs  law. 
obtained  results  of  far  more  practical  value  pertaining  to 
immunization.  He  established  the  fact  that  the  blood  and 
blood  serum  of  an  individual  which  has  been  artificially  ren- 
dered im??tune  against  a  certain  infectious  disease  may  be 
transferred  into  another  individual  with  the  effect  to  render  the 
latter  also  immune,  no  matter  how  susceptible  this  animal  is  to 
the  disease  in  question,       (Behring's  law.) 

This  discovery  became  fundamental  for  all  the  nu- 
merous investigations  which  hereafter  were  made  by  many 
German,  Italian  and  French  bacteriologists.  The  first 
announcement  concerning  the  artificial  immunity  against 
diphtheria  and  tetanus  by  blood  serum  was  published  by 
Behring  and  Kitasato,  in  the  Deutsch.  Medic,  Woch.,  i8go, 
in  which  they  declared  that  the  immunity  of  rabbits  and  mice 
when  rendered  immune  against  tetanus  is  based  upon  the  ability 
of  the  blood  serum  to  neutralize  the  toxins  produced  by  the 
tetanus  bacilli. 

Such  toxins  as  will  be  more  elaborately  explained 
under  the  chapter,  "Toxins  and  Toxalbumins,"  are  the 
poisonous  products  of  bacterial  metabolism,  and  are  the 
causes  for  acute  disease  when  circulating  in  the  organism. 
Their  effect  is  an  intoxication  of  the  system,  while  after 
introduction  of  virulent  germs  the  cause  of  the  disease  is 
an  infection.  In  this  case  the  microbes  multiply,  and  by 
change  of  products  form  a  poison  which,  when  absorbed, 
has  a  general  effect  as  a  secondary  result. 

In  the  mentioned  article  Behring  and  Kitasato  brought 
experimental  evidence  for  the  following  important  facts  :  1. 
The  blood  of  rabbits  immunized  against  tetanus  possesses 
the  ability  of  destroying  the  tetanus  toxin.     2.  This  ability 


12  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

remains  valid  when  the  blood  or  the  blood  serum  is  taken 
from  the  animal.  3.  By  transfusion  of  such  blood  serum 
into  other  animals,  the  latter  and  their  blood  is  rendered  also 
immune  against  tetanus.  4.  The  artificial  immunity  is  good 
not  only  against  inoculation  with  virulent  tetanus  bacilli, 
that  is  tetanus  infection,  but  also  against  tetanus  toxine, 
when  the  latter  as  a  chemical  poison  in  solution  is  injected. 
If  this  was  true,  the  natural  expectation  was  that  after  the 
toxin  had  already  poisoned  the  organism,  a  sufficient 
quantity  of  blood  serum  of  immunized  animals  would 
neutralize  the  circulating  toxin,  and  save  the  poisoned 
animal.  This  anticipation  was  thought  to  be  the  more 
correct  as  experiments  had  shown  that  wherever  the  toxin 
and  immunized  blood  serum  came  in  contact  even  in  the 
test  tube,  the  toxicity  of  the  former  was  neutralized  or 
completely  destroyed  by  the  antitoxic  properties  of  the 
latter.  Therefore,  the  experiment  to  inoculate  an  immune 
animal  with  the  toxine  was  now  reversed ;  that  is,  an 
animal  not  immunized,  but  suffering  from  tetanus  infection, 
was  inoculated  with  the  blood  serum,  and  the  effect  was 
recovery.  Thereby  the  first  evidence  was  established  for 
the  possibility  of  using  the  blood  serum  of  immunized 
animals  for  therapeutic  purposes. 

In  the  following  series  of  experiments  it  remained  to 
be  seen  how  the  serum  would  effect  man  in  case  of  infection 
or  intoxication.  It  was  found  that  in  every  instance  the 
above  mentioned  '^Behring's  Law"  held  good  ;  and  further, 
that  the  blood  serum  of  individuals,  naturally  immune 
against  a  certain  disease,  although  their  blood  was  resistant 
against  the  same,  does  not  possess  any  immunizing  prop- 
erties for  other  individuals.  This  very  important  fact 
proves  satisfactorily  that  the  immunizing  agent  is  not  a  sub- 
stance produced  by  nature  in  those  animals,  but  that  the 
production  of  it  is  a  result  of  an  organic  chemism,  to  which 


BLOOD  SERUM  THERAPY.  13 

the  impulse  must  be  given  by  introduction  of  the  corres- 
ponding poison. 

What  has  been  said  so  far  regarding  tetanus  applies 
as  well  to  diphtheria  and  similar  infectious  diseases.  The 
diphtheria  bacillus  also  produces  toxins,  the  effect  of 
which  is  intoxication  of  the  organism  which  can  be  bal- 
anced by  the  properly  prepared  antitoxin.  The  latter, 
first  introduced  by  Behring  and  Wernicke,  will  be  consid- 
ered at  length  under  the  chapter  "  diphtheria." 

In  accordance  with  the  above  mentioned  experiments,  immunization 

_^.     _,-,.,  11-  •  1  T->i'  against  vegeta- 

Prof.  Ehrlich  succeeded  in  showmg  that  the  "  Behrings  bie  poisons. 
Law  "  was  valid,  not  only  with  infection  or  intoxication  by 
bacterial  agents  but  also  for  some  purely  chemical  poisons, 
as  ricin,  an  albuminoid  present  in  the  seed  of  the  ricinus 
palm  possessing  extraordinary  toxicity  and  abrin,  the  tox- 
albumin  of  the  Jequirity-bean.  The  animals  immunized 
against  these  two  poisons  by  slowly  increased  doses,  fur- 
nished blood  serum  of  equally  immunizing  properties  for  the 
corresponding  poison  as  was  shown  in  tetanus  and  diph- 
theria. The  gradual  increased  dose  of  these  poisons  ren- 
dered the  animals  more  and  more  immune  until  a  certain 
degree  of  immunity  had  been  reached.  That  this  gradual- 
ly increasing  immunity  was  not  a  simple  tolerance  of  the 
poison  was  proved  by  the  fact,  that  the  blood  of  im- 
munized animals  contained  an  antitoxic  substance  called 
antiricine  and  antiabrin,  which,  added  to  the  poison  itself, 
would  attenuate  and  even  neutralize  the  latter's  toxicity. 
The  results  obtained  in  tetanus  and  diphtheria,  were  also 
obtained  in  other  diseases,  such  as  different  forms  of  sep- 
ticaemia and  even  in  hydrophobia,  the  bacterial  cause  of 
which  is  as  yet  unknown.  In  these  particular  diseases  it 
was  found  as  early  as  1889,  by  Babes  and  Sepp,  that  the 
blood  serum  of  immunized  individuals,  injected  into  others 
rendered  the  latter  also  immune.     The  immediate  effect  of 


> 


14  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

these  remarkable  discoveries,  was  a  better  understanding 
as  to  the  spontaneous  recovery  from  infectious  diseases. 
It  seems  that  in  all  of  them  the  blood  produces  certain 
substances,  antagonistic  to  the  toxins,  which  had  caused 
the  disease,  and  after  the  poison  had  been  neutralized  the 
residual  antitoxins  afford  such  immunity  as  is  generally 
observed  in  scarlet,  measles,  smallpox,  etc.  This  theory 
is  substantiated  by  recent  reports  that  blood  serum  of  pa- 
tients, who  had  recovered  from  pneumonia,  typhoid  and 
cholera,  possessed  immunizing  power  to  animals. 

Having  thus  succeeded  in  securing  a  method  to  pre- 
vent, as,  under  favorable  conditions,  also  to  cure  persons 
from  infectious  diseases,  it  was  of  material  importance  to 
find: 

1st,  a  mode  of  estimating  the  strength  of  immunizing 
serum,  and, 

2d,  the  best  way  to  procure  the  material  of  highest 
immunizing  power.  Both  problems  were  satisfactorily 
solved  by  Behring,  Wernicke,  Boehr  and  Kossel,  whose 
experiments  will  be  given  consideration  later.  In  order  to 
secure  the  highest  possible  strength,  one  has  to  remember 
that  the  value  of  the  serum  depends  on  the  relative  degree 
of  immunity,  that  is,  the  difference  between  the  pre- 
vious susceptibility  and  the  acquired  immunity.  For  this 
reason  it  would  be  proper  to  take  the  serum  of  a  very 
susceptible  animal  which  has  been  made  resistant,  but  still 
reacts  to  inoculation  of  the  poison.  The  degree  of  immun- 
ity is  determined  by  the  figure  which  indicates  how  many 
times  the  minimum  dose,  necessary  to  kill  an  animal  of 
equal  weight,  may  be  multiplied  without  fatal  effect.  The 
immunizing  value,  however,  is  given  by  the  figure  indicat- 
ing how  much  animal  weight  in  grammes  can  be  protected 
by  one  gramme  of  serum,  if  the  inoculation  of  the  toxin 
has  taken   place  twenty-four  hours  after  the  application  of 


BLOOD  SERUM  THERAPY.  15 

the  serum.  This  calculation  must  necessarily  be  taken  into 
consideration  as  it  requires  a  certain  length  of  time  before 
the  animal  enjoys  the  full  benefit  of  the  immunizing  ma- 
terial. In  this  or  similar  conditions,  which  means,  as  long 
as  the  application  of  serum  is  made  previous  to  the  inocu- 
lation, the  quantity  of  serum  required  to  prevent  the  dis- 
ease, is  much  smaller  than  after  the  inoculation,  and,  while 
in  the  former  case  1  grain  of  serum  of  a  standard  strength 
called  *' Normal  therapeutic  serum  "  is  sufficient,  it  may  re- 
quire 500  such  doses  to  save  an  animal  but  twenty-four 
hours  after  the  intoxication. 


CHAPTER  II. 


Toxins  and  Toxalbumins. 

The  above  statements  may  be  sufficient  to  outline  the 
nature  and  principles  of  blood  serum  therapy  in  general 
and  in  the  following  we  will  have  to  deal  with  the  details 
of  this  promising  discovery  of  modern  medicine.  Being 
confronted  in  every  phase  of  serum  therapy  with  bacterial 
poisons  the  character  of  which  has  up  to  late  been  un- 
known, it  seems  to  be  proper  to  pay  especial  attention  to 
these  agents,  which  are  of  so  material  importance  in  de- 
tecting the  problem  of  infection  and  its  cure,  before  going 
into  particulars  of  the  therapeutic  measures. 

After  it  had  become  an  assured  fact  that  tetanus  is 
transferable  by  inoculation  of  the  fluid  discharged  from  the 
original  wound,  Prof.  Brieger  succeeded  (1880)  by  his  in- 
genious investigations  on  ptomains  and  toxins  to  prove 
that  a  certain  crystalline  substance  of  great  toxicity  can  be 
isolated  from  tetanic  fluids  and,  when  injected  into  animals 
would  reproduce  tetanus.  The  existence  of  this  substance, 
called  tetanin,  was  obtained  not  only  from  animals  but 
also  from  the  arm  of  an  infected  patient,  a  fact  which  ren- 
dered sufficient  evidence  that  such  toxins  are  present  in 
the  mentioned  disease.  This  remarkably  discovery  was 
immediately  followed  by  Kitasato's  equally  valuable  suc- 
cess in  securing  pure  cultures  of  the  tetanus  bacillus. 

There  was  good  reason  to  believe  that  other  microbes 
produce  similar  substances  and  indeed  quite  a  number  of 
toxins  were  revealed. 

The  preparation  of  pure  cultures  was  in  itself  a  war- 


TOXINS  AND   TOXALBUMINS.  17 

rant  for  further  success  in  obtaining  the  chemical  material 
which  is  essential  in  transferring  infectious  disease,  and 
such  substances  were  soon  discovered  in  typhoid,  cholera 
and  other  pathogen  microbes.  The  effect,  obtained  by  in- 
oculation of  the  so  prepared  toxins  was  not  always  identical 
with  the  symptoms  of  the  genuine  disease,  neither  were 
the  toxins  present  in  every  culture  not  even  of  the  better 
known  microbes.  It  was  therefore  natural  to  search  for 
the  factors  which  were  causing  this  inconsistency  of  affairs 
and  to  ascertain  whether  the  disease  depended  on  still  other 
products.  Brieger  and  Fraenkel,  who  vigorously  investi- 
gated these  questions,  recognized  the  difficulty  which 
existed,  as  long  as  they  could  not  separate  the  microbes 
from  their  toxic  products  and  commenced  to  experiment 
with  such  cultures  in  which  the  living  microbes  were  de- 
stroyed by  heat.  But  they  soon  found  that  with  the  death 
of  the  microbes  the  whole  culture  became  ineffective  and 
so  had  to  employ  another  more  reliable  method.  This  was 
found  in  using  a  Chamberland  or  Choalin  filter  which  per- 
mitted the  liquid  substrate  of  the  culture  to  pass  while  the 
microbes  of  almost  every  species  were  kept  back  by  the 
filter.  It  was  about  at  this  time  (1884)  when,  as  a  result  Loeffler's 
of  many  experimental  and  bacteriological  researches,  Prof. 
Loeffier  found  a  bacillus  which  was  considered  the  cause 
for  diphtheria  and  being  an  extraordinary  good  subject  for 
their  study,  it  was  this  microbe  on  which  Brieger  and 
Fraenkel  continued  their  work  in  a  systematic  way.  Soon 
however,  they  were  confronted  with  another  problem.  The 
cultures,  usually  kept  in  an  incubator  for  four  to  six  or  eight 
weeks  showed  when  transferred  upon  another  nourishing 
substrate  a  different  degree  of  toxicity  and  sometimes  so 
much  less  that  beyond  doubt  a  change  had  taken  place  in 
these  cultures.  This  gave  reason  to  believe  that  their  toxic- 
ity was  due   to   other  than  such  basic  substances  as  repre- 


18  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

sented  by  toxins  and  ptomains.  Both  investigators  came 
to  the  conclusion,  in  accordance  with  Roux  and  Yersin,  of 
Paris,  who  simultaneously  worked  in  the  same  line,  that  the 
toxic  substances  produced  by  the  microbes  must  be  of  al- 
buminous nature,  and  after  numerous  experiments  they  suc- 
ceeded in  preparing  such  toxic  albuminoids  which  they 
called  "toxalbumins."  These  substances  are  of  different 
nature  than  the  toxins  and  cannot  be  crystallized,  but 
represent  an  amorphic  substance.  They  were  always  found 
not  only  in  the  cultures  but  also  in  the  diseased  organism 
and  showed  at  times  an  exceptionally  high  degree  of 
toxicity. 
Nature  and  Being  without    doubt    derivatives  of  the   albumen   of 

preparation  of  '  .  r      i      •  i      • 

tpxaibumins.     tissue  they  are  on  account  of   their  relation  to  the  latter  of 
\  much  more  pathological  interest.     It  was    thereby  proved 

that  the  metabolism  of  normal  substance  can  produce  ma-, 
terial  of  most  detrimental  character  by  means  of  bacterial 
action.  The  important  influence  of  the  toxalbumins 
upon  the  organism  was  more  positively  demonstrated  by 
the  effect  of  the  toxalbumin  of  anthrax,  being  the  first 
prepared  as  a  pure  chemical  by  Brieger  and  Fraenkel,  from 
the  organs  of  a  diseased  rabbit,  which  had  been  inoculated 
with  anthrax  bacilli.  The  animal,  treated  with  this  toxal- 
bumin, showed  all  symptoms  of  the  original  disease.  The 
corresponding  experiment  was  made  by  Dr.  Immerwahr 
with  equal  result  in  tetanus.  The  way  of  proceeding  was 
as  follows:  the  heart,  spleen,  kidneys  and  liver  of  an  ani- 
mal, just  perished  after  inoculation  with  Kitasato's  pure 
tetanus  culture,  were  cut  under  aseptic  precautions  into 
small  pieces  and  mixed  with  six  ounces  of  distilled  water. 
The  filtered  extract  was  prepared  as  advised  by  Brieger 
and  Fraenkel  until  the  amorphic  substance  was  obtained. 
A  solution  of  the  latter,  sterilized  by  filtration  through  a 
Chaolin  filter,  was  in  as  small  quantities  as   0.005  grm.  in- 


No.  1.     Anthrax  Bacilli. 

The  specimen  is  taken  from  the  spleen  juice  of  a  guinea  pig  infected 
with  anthrax  ;  stained  in  gentian  violet,  magnified  500  times,  condensor. 

Besides  cellular  elements  it  contains  the  anthrax  bacilli,  surrounded 
by  a  membrane  which  appears  as  a  capsule  with  the  bacillus  as  nucleus. 
The  anthrax  affection  has  to  be  considered  a  genuine  septicaemia,  the 
infection  progressing  along  the  course  of  the  circulation.  For  this  rea- 
son the  bacilli  are  found  principally  in  the  smaller  veins  and  capillary 
system. 


TOXINS  AND  TOXALBUMINS.  19 

jected  into  mice  which  after  twenty  hours  showed  the  com- 
mon symptoms  of  tetanus  and  died  within  forty-eight 
hours. 

From  the  positive  result  of  these  experiments 
Immerwahr  concluded,  that  similar  toxalbumins  may  pos- 
sibly be  obtained  even  in  diseases,  the  bacterial  cause  of 
which  has  not  yet  been  established.  He  therefore  experi- 
mented with  the  blood,  taken  from  a  patient  who  suffered 
from  a  severe  form  of  scarlet  fever;  however,  the  effort  to 
secure  the  chemical  preparation,  or  the  inoculation  of  such 
blood  to  mice  was  of  no  positive  result.  In  another  case 
of  scarlet  fever  combined  with  uraemia  he  could  not  obtain 
a  substance,  charateristic  to  scarlet  but  succeeded  in  pre- 
paring an  albuminoid  which  inoculated  to  animals  caused 
severe  spasms  and  finally  death,  therefore  was  considered 
responsible  for  the  uraemia.  Returning  to  the  experiments 
with  toxalbumins  of  tetanus  and  diphtheria  Brieger  as 
well  as  Immerwahr  and  others  met  with  peculiar  experi- 
ence, as  they  frequently  observed  a  difference  in  the  inten- 
sity of  the  respective  toxalbumins. 

The  same   phenomenon    had   been  observed  by  Roux  variability  of 

1  xr         •  •  f  toxicity. 

and  Yersm  with  reference  to  the  toxicity  of  pure  bacterial 
cultures;  besides  this  a  series  of  other  questions  remained 
still  unanswered  :  Why  is  the  toxic  effect  of  these  toxalbu- 
mins so  slow  compared  to  that  of  other  strong  poisons? 
In  what  direction  do  they  act  within  the  diseased  organism? 
What  makes  the  latter  immune  against  an  infectious  dis- 
ease after  having  recovered  from  same? 

During  the  further  study  of  the  subject  some  impor- 
tant items  were  noticed  which  led  the  experimenters  upon 
the  right  track  in  answering  such  questions.  They  found 
that  pure  cultures  in  beef  juice  did  not  retain  their  original 
toxicity  for  a  longer  period  but  became  attenuated  and  at 
last  ineffective  even  when  used  in  large  quantities.     In  ac- 


20  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

cordance  with  Brieger  and  Fraenkel  two  other  investigators 
— Wassermann  and  Proskauer — found  in  1891  that  the  cul- 
tures of  diphtheria  bacilli  lost  their  virulence  when  trans- 
planted from  beef  juice  upon  glycerin-agar,  that  they  could 
however  regain  part  of  it  as  Roux  and  Yersin  demon- 
strated, when  brought  back  into  beef  juice. 
Discovery  of  ^t    the  Same    time    they    discovered    in    the    cultures 

nonpoisonous  -' 

albumins.  bcsidcs  the  toxalbumiu  a  second  substance  of  albuminous 
nature,  but  different  from  the  former  in  chemical  as 
well  as  physiological  respect.  The  new  substance  was 
soluble  in  diluted  alcohol  and  when  injected  into  animals 
proved  to  be  perfectly  harmless.  In  order  to  reveal  the 
characteristic  properties  of  both  substances  it  was  neces- 
sary to  separate  them  from  each  other,  and  this  was  effected 
by  the  following  process:  The  cultures  in  beef  juice  are 
filtered  through  Choalin  and  the  fluid,  now  free  from  bacte- 
rial cells,  is  condensed  in  vacuo  to  one-tenth  of  its  volume, 
and  for  three  days  kept  in  the  dialyzer  with  distilled  water,  so 
as  to  separate  the  peptones  and  globulins,  then  again  fil- 
tered until  perfectly  clear  it  is  mixed  with  ten  times  its 
volume  of  slightly  sour  alcohol  (60-70  per  cent).  The 
now  appearing  precipitate  remains  in  the  solution  for  forty- 
eight  hours,  which  after  being  filtered  is  then  slowly 
dropped  into  absolute  alcohol  in  which  a  new  precipitate 
appears.  Both  precipitates  are  collected  and  exammed 
separately.  The  toxalbumin,  after  having  gone  through 
the  usual  process  of  dialyzation,  etc  ,  appears  as  a 
fine  white  amorphic  substance,  the  other  precipitate  as  a 
more  yellowish  brown.  The  difference  between  these  two 
substances  is  most  evident  with  reference  to  their  physio- 
logical action.  The  toxalbumin  possesses  a  high  degree 
of  toxicity.  The  yellow  precipitate  has  none  at  all.  In  ac- 
cordance herewith  Proskauer  and  Wassermann  found  that 
very   virulent    cultures    contained    a    proportionally   large 


No.   2.     Anthrax  Bacilli. 

Section  of  the  liver  of  a  guinea  pig  infected  with  anthrax  ; 
stained  in  fuchsin  solution,  magnified  500  times. 

The  tissue  is  not  visibly  affected,  especially  not  necrotized.  The 
microbes  are  located  within  the  blood  vessels.  A  small  vein  and  capil- 
lary are  represented  in  the  specimen  filled  with  bacilli.  In  pure  cul- 
tures the  latter  form  long  chains,  leaving  a  small  space  between  two 
links  which  is  probably  due  to  a  shrinkage,  or  contraction  of  the 
membrane  mentioned  above. 


TOXINS  AND  TOXALBUMINS.  21 

quantity  of  the  white  substance  but  little  of  the  yellow,  and 
the  ineffective  cultures  more  of  the  yellow  than  of  the 
white  substance. 

The  experiments  made  on  animals  had  the  follow- 
ing results:  one-eighth  of  a  grain  of  the  white  sub- 
stance, injected  into  a  rabbit,  killed  the  latter  in  three 
or  four  days,  one-sixteenth  grains  in  two  weeks,  one- 
twentieth  in  two  months.  The  intensity  of  toxalbumin, 
however,  was  not  steady,  and  showed  considerable  differ- 
ence even  if  the  Original  culture  of  which  it  had  been  pre- 
pared had  not  changed  its  virulence.  For  this  reason  it 
was  impossible  to  establish  the  minimum  fatal  dose.  More 
remarkable,  however,  is  the  fact  that  notwithstanding  the 
extraordinary  small  quantity  necessary  for  fatal  result,  the 
poison  has  an  unusual  slow  effect.  The  different  physio- 
logical actions  of  the  two  substances  obtained,  led  to  the 
idea  that  a  certain  relation  may  exist  between  them,  within 
the  infected  living  organism,  and  starting  from  this  point 
of  view  the  above  named  experimenters  tried  to  isolate 
them  from  the  organs  of  animals  which  had  died  from 
diphtheria. 

As  before,  for  the  preparation  of  the  toxalbumins,  the 
lungs,  kidneys,  liver  and  spleen  were  extracted  and  sub- 
mitted to  a  similar  process  as  mentioned  above.  These 
experiments,  however,  were  negative  as  far  as  the  prepara- 
tion of  the  second  yellow  substance  is  concerned,  and  the 
toxalbumin  in  this  way  prepared  proved  to  be  still  more 
effective  than  the  one  obtained  from  pure  cultures. 
^i^  grm.  was  sufficient  to  kill  an  animal  in  one 
to  two  weeks.  The  pathological  changes  observed  in 
such  animals  by  a  post  mortem  examination  were  corre- 
spondent with  the  usual  organic  symptoms  in  diphtheria, 
except  the  local  inflammation  as  caused  by  infection  of  vir- 
ulent cultures.      Further  experiments,  intending  to  show  a 


22  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

relation  between  these  two  substances  and  the  artificial 
immunization  of  animals  against  diphtheria  were  at  that 
time  also  negative,  and  it  took  several  years  before  this 
question  could  be  answered  more  satisfactorily.  The  facts 
so  far  obtained  and  proved  by  experiments  were  : 

1.  Infectious  diseases  are  transferred  by  their  specific 
microbes  and  the  products  of  the  latter. 

2.  By  the  action  of  the  microbes  toxic  substances  are 
produced  which  are  the  essential  cause  for  the  disease. 

3.  These  toxic  substances,  called  toxalbumins,  are  of 
albuminous  nature,  but  have  no  steady  degree  of  toxicity. 

4.  The  toxalbumins  can  be  prepared  as  well  from 
cultures  from  the  microbes  as  from  parts  of  a  diseased 
organism  in  which  they  are  circulating. 

5.  The  inoculation  with  either  the  blood  serum  of  an 
infected  animal  or  with  the  toxalbumins  brings  on  similar 
pathological  conditions  as  in  the  original  case. 

6.  The  existence  of  another  substance,  antagonistic  to 
toxalbumin  in  its  physiological  effect. 

It  was  especially  the  last  mentioned  substance,  the  true 
nature  of  which  was  then  studied  with  deepest  interest,  as 
it  was  considered  instrumental  for  artificial  immunity. 
Among  those  who  tried  to  explain  this  phenomenon  Prof. 
Ehrlich,  of  Berlin,  ranked  first.  Recognizing  the  difficulty 
of  experimenting  with  a  pure  toxalbumin  prepared  from 
microbes  or  diseased  organs,  he  selected  the  toxic  albumi- 
noids of  two  vegetable  products — ricin,  of  the  seed  of  the 
ricinus  palm,  and  abrin,  of  the  jequirity  bean,  both  of  which 
were  at  his  disposal  in  sufficient  quantity  and  purity.  He 
succeeded  in  not  only  immunizing  animals  against  these 
strong  poisons,  but  also  in  determining  by  what  method  to 
obtain  the  highest  degree  of  immunity.  In  some  animals 
he  applied  hypodermic  injections,  others  were  fed  with  the 
tox?lbumin,  and  in  some  the  application  of  a  drop  of  its 


TOXINS  AND  TOXALBUMINS.  23 

solution  to  the  conjunctiva  was  sufficient  for  a  positive 
result.  He  further  proved  that  the  immunity  obtained  by 
application  of  gradually  increased  doses  of  the  toxalbumin 
is  not  identical  with  a  mere  tolerance  toward  the  poison, 
and  has  a  certain  limit  beyond  which  the  resistance  against 
the  toxic  substance  cannot  be  increased.  For  instance,  a 
mouse  made  immune  against  200  times  the  fatal  dose  died 
when  given  an  additional  dose  of  the  poison,  in  spite  of 
being  continually  fed  with  small  quantities. 

In  the  meantime  Behring  and  Kitasato  made  the  funda- 
mental discovery  that  the  immunity  in  diphtheria  and  teta- 
nus was  based  upon  a  certain  ability  of  the  blood  to  neu- 
tralize the  toxicity  of  bacterial  poisons,  and  this  led  Ehrlich 
to  the  idea  that  the  immunity  against  such  toxalbumins  as 
he  found  in  vegetable  products  was  due  to  a  well  character- 
ized substance  in  the  blood  itself.  He  at  last  found  that 
indeed  such  a  substance  existed,  which  he  called  "anti- 
ricin,"  the  injection  of  which  to  mice  and  rabbits  rendered 
the  ricin  ineffective  and  the  animals  immune.  It  was  this 
most  important  discovery  which  became  the  basis  for  all 
the  now  following  researches  of  the  investigators  intending 
to  find  in  the  various  infectious  diseases  the  analagous  sub- 
stances hereafter  called  antitoxins.  Behring  and  Ehrlich 
next  established  the  fact  that  the  injection  of  blood  taken 
from  immune  animals  rendered  others  also  immune.  It  was 
a  priori  evident  that  the  degree  of  immunity  of  an  animal 
depended  on  the  amount  of  antitoxin  present  in  the  blood 
injected.  Further  experiments  intended  to  determine  the 
quantitative  estimation  of  the  antitoxins,  and  the  duration 
as  well  as  the  degree  of  immunity,  opened  a  wide  perspec- 
tive for  this  new  and  rapidly  progressing  science.  Having 
explained  how  the  improved  knowledge  of  the  aetiology  of 
infectious  diseases  led  to  more  appropriate  therapeutic 
measures  than  were  known  heretofore,  we  will  now  con- 


24  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

siderin  detail  what  uses  were  made  of  the  above-mentioned 
discoveries. 

The  two  diseases  in  reference  to  which  the  great- 
est number  of  experiments  were  made,  and  in  which 
the  most  important  results  were  obtained,  are  tetanus  and 
diphtheria.  The  results  accomplished  is  best  demonstrated 
by  quoting  the  experiments  of  the  various  investigators. 


CHAPTER  III. 


Tetanus. 

The  first  proof  that  tetanus  is  an  infectious  disease 
was  presented  by  Carle  and  Rattone  in  1884,  who  inocu- 
lated a  rabbit  with  the  pus  taken  from  a  tetanus  wound 
of  a  man  and  thereby  reproduced  the  disease  in  the  rabbit. 
Soon  after  Dr.  Nicolaier,  engaged  in  examining  the  mi- 
crobes of  soil,  frequently  met  with  the  same  bacillus,  and  bacfnus. 
observed  the  strange  fact  that  if  some  of  the  soil  contain- 
ing the  latter  were  applied  hypodermically  to  guinea  pigs 
and  mice,  it  caused  spasms  after  one  to  five  days  ;  first 
locally,  later  in  other  groups  of  muscles.  In  the  pus,  dis- 
charged from  the  infected  part,  he  found  in  every  case  a 
long  straight  bacillus.  A  similar  microbe  was  seen  by 
Rosenbach  in  cases  of  human  tetanus,  but  neither  one  of 
the  investigators  succeeded  in  finding  these  bacilli  any- 
where except  in  the  nearest  vicinity  of  the  place  of 
infection,  and  the  experiment  to  cultivate  them  from  the 
blood  or  organs  was  also  negative.  The  violent  effect  of 
these  microbes,  without  increasing  much  in  number  nor 
spreading  over  the  organism,  led  to  the  conclusion  that 
they  produce  a  strong  poison,  and  as  mentioned  above, 
this  was  corroborated  by  Brieger's  preparation  of  a  sub- 
stance from  cultures,  tetanin,  which  when  inoculated 
into  animals  reproduced  the  disease.  He  also  proved  by 
preparing  this  tetanus  poison  from  an  amputated  arm  of  a 
patient,  that  after  an  infection  it  circulates  in  the  system. 
At  last  Kitasato  found  a  method  to  prepare  pure  cultures 
of   the    microbe    and    thereby     furnished    unquestionable 


26  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

evidence  of  the  bacterial  nature  of  the  disease.  Further 
experiments  made  at  the  surgical  clinic  at  Halle,  in  1891, 
by  Dr.  Nissen  with  the  blood  serum  of  a  patient  suffering 
of  tetanus,  showed  that  mice,  injected  with  very  small 
quantities  of  such  serum,  died  within  a  few  hours  under 
symptoms  identical  with  those  in  the  original  case. 

The  knowledge  of  circulation  of  toxins  in  the  blood 
which  are  the  products  of  microbes,  caused  naturally  the  de- 
sire to  nullify  their  effect  by  antagonistic  substances,  /.  e,, 
antitoxins,  and  like  the  administration  of  bactericide  sub- 
stances in  antisepsis,  it  now  manifested  itself  a  tendency 
to  find  the  proper  antitoxic  material.  The  bactericide 
substances  have  on  account  of  this  theory  not  lost  any  of 
their  importance,  remaining  as  necessary  as  ever  for  pre- 
venting infection,  sterilizing  instruments,  etc.,  but  in  the 
living  organism  they  are  worthless,  at  least  for  the  ma- 
jority of  cases. 

In  the  administration  of  antitoxins  our  aim  is  not 
so  much  to  kill  the  microbes  but  to  paralize  the  poison 
produced  by  them,  knowing  that,  after  this  has  been 
effected,  most  any  bacterial  cell  is  comparatively  harm- 
less as  far  as  the  immunized  individual  is  concerned. 
The  cholera  bacilli,  for  instance,  which  are  daily  dis- 
charged from  the  intestines  of  a  convalescent  are  innocu- 
ous for  him  because  of  his  acquired  immunity. 
Immunity.  With  reference  to  the  nature  of   immunity  we  have  to 

distinguish  two  entirely  different  conditions  not  only  from 
an  aetiological  standpoint,  but  also  in  regard  to  their  thera- 
peutic value.  1.  The  protections  against  certain  bacterial 
/  poisons  may  be  natural.  In  this  case  the  natural  immunity 
is  caused  by  the  lack  of  susceptibility  to  the  poisons  of 
such  organs  as  are  affected  in  susceptible  individuals. 
The  immunity  is  therefore  hereditary  and  has  no  relation 
to  the  composition  of  the  blood,  consequently  it  cannot  be 


No.  3.     Tetanus  Bacilli. 

Culture  on  agar;  stained  in  fuchsin  solution,  magnified  1,000  times. 

The  pathogen  microbe  in  tetanus  is  a  slender  bacillus  with  distinct 
self-motion.  Cultivated  in  higher  temperature  the  germs  soon  produce 
spores,  appearing  on  their  ends  and  thereby  giving  them  the  shape  of  a 
drumstick.  Tetanus  bacilli  are  strictly  anaerobe  microbes,  and  when 
transferred  upon  gelatin,  form  in  the  lower  part  of  the  substrate  charac- 
teristic colonies  with  numerous  branches,  gradually  liquefiying  the 
gelatin. 


TETANUS,  27 

transferred  upon  another  individual  by  the  blood  serum. 
Such  immunity  is  possessed  by  white  mice  against  diph- 
theria, and  chickens  against  tetanus.  2.  In  contrast  to  this 
is  the    acquired    immunity  in    susceptible  individuals  ac-  / 

complished  by  application  of  gradually  increased  doses  of  a 
certain  poison  or  by  passing  through  the  corresponding 
sickness,  as  has  been  demonstrated  in  a  horse  which  is 
naturally  very  susceptible  to  tetanus,  it  being  able  to  stand 
several  million  times  as  much  of  the  tetanus  toxin  as  would 
kill  another  horse,  not  treated  before.  By  such  systematic 
applications  of  the  poison,  the  blood  serum  acquires  the 
ability  to  neutralize  its  effect,  which  means  ;  (a)  a  chemi- 
cal change  of  its  composition  ;  (b)  a  decreased  reaction  of 
living  tissue  upon  tetanus — intoxication.  The  effect  of  Effect  of 
tetanus  poison  to  susceptible  individuals  is  therefore  a 
threefold  one  : 

(1).  The  symptoms  of  a  disease  peculiar  to  tetanus. 

(2).  The  production  of  tetanus  antitoxin  in  the  blood. 

(3).   The  modification  of  susceptibility  of  the  elements 
to  the  tetanus  toxin. 

The  symptoms  of  tetanus  are  well  known  and  will 
find  their  explanation  with  the  last-named  effect.  The 
latter,  as  well  as  the  production  of  antitoxin  has  been 
the  subject  of  deepest  study  on  the  part  of  several 
investigators,  the  result  of  which  may  be  explicated  in  the 
following.  A  remarkable  phenomenon,  first  commented  on 
by  Behring,  is  that  the  immunity  of  animals  does  not  de- of  elements  of 
pend  upon  the  presence  of  antitoxin  in  the  blood  but  can 
remain  even  if  no  antitoxin,  which  gradually  disappears 
from  the  organism,  can  be  traced  any  more  in  the  serum. 
In  other  words,  the  resistance  against  the  poison  and  the 
amount  of  antitoxin  present,  are  not  corresponding.  Some 
sheep  immunized  against  tetanus,  possessed  a  higher  de- 
gree of  immunity  after  all  antitoxin  had  disappeared  from 


28  BLOOD   SERUM  THERAPY  AND  ANTITOXINS. 

their  blood,  than  when  it  contained  the  largest  quantity  of 
antitoxin.  In  such  a  case  the  condition  of  an  animal  can 
become  identical  with  one  that  is  naturally  immune,  that  is, 
its  immunity  can  increase  to  such  an  extent  that  an  innoc- 
ulation  with  even  the  most  virulent  culture  has  no  effect, 
while  the  blood  is  free  from  antitoxins.  Under  these  cir- 
cumstances, however,  the  animal  is  unfit  to  have  its  immu- 
nity transferred  upon  others  by  inoculation  with  the  serum. 
It  was  hereby  shown  that  the  presence  of  antitoxin  alone 
does  not  only  explain  the  immunity  obtained  by  application 
of  the  poison  but  that  certain  elements  of  the  organism, 
which  before  were  susceptible  to  the  disease,  have  become 
insusceptible. 
^iveTmmuiSry.'  Different    from    the  condition   brought    on    by    inoc- 

ulation with  poison,  which  is  an  indirect  immuniza- 
tion, is  the  direct  immunization  by  application'  of  tlie 
antitoxin.  Individuals  so  immunized  remain  immune 
only  as  long  as  their  blood  contains  antitoxin  and  with 
disappearance  of  the  latter  they  become  as  susceptible 
again  as  they  were  before.  It  is  necessary  to  distinguish 
these  two  conditions  clearly,  the  antitoxin  immunity  (pas- 
sive immunity,  Ehrlich)  being  based  upon  a  temporary 
addition  of  a  protecting  agent  to  the  blood,  and  the  im- 
munity of  the  elements  being  caused  by  an  intracellular 
transformation  to  the  effect  of  lasting  insusceptibility.  The 
relation  between  these  two  different  kinds  of  immunity  has 
been  carefully  studied  by  Behring  who,  in  connection  with 
this,  established  the  fact  that  during  the  treatment  with 
toxins,  animals  may  acquire  even  an  increased  suscepti- 
bility instead  of  getting  immune,  and  at  the  same  time  pos- 
sess a  large  amount  of  antitoxin  in  their  blood.  Before 
this  was  known  a  large  quantity  of  antitoxin,  found  in  the 
serum,  was  considered  evidence  for  the  immunity  of  the 
respective    animals.     Hereafter,    however,    it    was    shown 


TETANUS.  29 

that  notwithstanding  the  production  of  antitoxin  in  the 
blood,  an  animal  may  not  only  be  not  immune  but  more 
susceptible  than  before.  Speaking  on  this  subject,  Behr- 
ing  mentions  a  horse  the  blood  of  which  contained  in  one 
ccm.  enough  tetanus  antitoxin  to  immunize  other  horses 
against  such  a  dose  of  tetanus  toxin,  a  small  fraction  of 
which  would  be  sufficient  to  kill  the  former.  The  knowl- 
edge of  this  fact  is  of  great  practical  value  for  the  prepar- 
ation of  therapeutic  serum,  as  it  shows  that  the  animals 
most  fit  to  furnish  a  good  therapeutic  serum  are  not  those 
that  have  the  highest  degree  of  immunity  but  those  in 
whom  the  inoculation  of  toxin  is  followed  by  a  most 
marked  reaction. 

The  following  report  of  some  cases  of  human  tetanus 
may  demonstrate  the  results  obtained  in  this  disease  by 
means  of  serum  therapy. 

The  first  case  of  human  tetanus  treated  with  antitox- 
in has  been  reported  by  Dr.  Gagliardi,  of  Mollinelli, 
province  of  Bologna.  The  antitoxin  used  in  this  case 
was  prepared  in  the  laboratory  of  Prof.  Guido  Tizzoni 
and  Cattani,  two  of  the  most  industrious  investigators 
of  this  subject.  The  patient,  a  man  forty-five  years  of 
age,  accidentally  contracted  a  wound  on  the  small  toe  of  the 
left  foot,  while  going  over  a  rice  field.  On  the  next  day, 
May  12,  1891,  he  consulted  Dr.  Gagliardi,  as  the  foot  had 
swollen  considerably.  The  Doctor  made  an  incision  and 
treated  the  wound  with  antiseptics.  On  May  19,  it  was 
healed.  Four  days  later  the  patient  had  symptoms  of 
trismus,  which  became  very  pronounced  on  May  24.  In- 
jections of  5  per  cent  carbolic  acid  in  the  vicinity  of  the 
wound  were  of  no  effect.  On  June  3,  opisthotonus,  and  all 
symptoms  increasing.  At  this  time  the  first  dose,  0.25 
ccm.  of  Tizzoni's  antitoxin,  obtained  from  a  strongly  im- 
munized dog,  was  injected,  after  which  some  improvement 


30  BLOOD   SERUM  THERAPY  AND  ANTITOXINS. 

was  observed.  On  June  7,  after  relapse  and  general 
tetanic  spasms,  two  more  injections  were  made.  The  fol- 
lowing day  the  patient  gradually  improved  and  was  dis- 
charged as  cured  on  July  5.  In  this  case  less  than  1 
ccm.  of  antitoxin  was  sufficient  to  neuralize  the  tetanus 
toxin. 

Another  case  was  reported  by  Dr.  Swartz,  December, 
1891.  The  patient,  a  boy  of  fifteen  years,  cut  himself  on 
the  left  forearm,  August  20,  1891,  while  opening  a  walnut 
which  he  had  picked  up  from  the  ground.  Fourteen  days 
later  he  noticed  contractions  of  the  muscles  of  the  arm,  and 
during  the  two  following  days  also  in  both  legs  and  the  jaw. 
Applications  of  chloral  and  warm  baths  were  of  no  effect. 
On  September  18,  Prof.  Tizzoni  injected  0.15  ccm.  of  dog 
antitoxin,  which  he  repeated  twice  on  the  next  day.  Here- 
after the  patient  showed  signs  of  improvement.  The  in- 
jections were  repeated  on  the  three  following  days  with 
0.25  ccm.  antitoxin  at  each  time.  On  September  23  the 
symptoms  of  tetanus  had  disappeared  and  on  October  1 
the  patient  could  leave  the  hospital. 

Early  in  1892  Dr.  E.  Pacini  reported  the  third  case 
treated  successfully  with  antitoxin.  A  peasant,  twenty- 
one  years  of  age,  cut  himself  with  a  sickle  on  the  left  hand, 
October  25,  1891.  November  4  he  had  difficulty  in  opening 
the  mouth,  suffered  from  neuralgic  pains  and  general  weak- 
mess.  November  13,  trismus,  but  no  tetanus  of  the  limbs. 
During  the  next  six  days  the  symptoms  spread  over  to 
other  muscles  and  November  19  almost  general  tetanus  set 
in.  After  two  injections  of  0.25  ccm.  of  antitoxin  for  four 
consecutive  days  patient  began  to  improve,  but  was  not 
quite  free  from  tetanus  symptoms  until  December  15. 
During  all  this  time  the  muscles  of  the  limbs  had  not  been 
affected. 

The  following  case,  also  resulting  in  recovery,  was  re- 


TETANUS.  31 

ported  by  Dr.  G.  Taruffi  in  La  Riforma  Med.,  1892.  A 
peasant,  seventy-four  years  of  age,  while  loading  a  wagon 
with  wood,  severely  lacerated  his  right  hand.  Ten  days  later 
the  first  symptoms  of  tetanus  appeared  and  increased  rap- 
idly during  the  next  two  days.  He  at  once  received  an 
injection  of  0.25  ccm.of  antitoxin,  which  caused  slight  im- 
provement and  intense  perspiration.  The  injections  were 
repeated  daily  in  the  forenoon  and  afternoon  until  he  had 
received  six  doses,  each  time  with  the  same  result.  His 
urine  taken  before  the  first  injection  and  injected  into  mice 
had  a  fatal  effect  within  twenty-four  hours;  the  urine,  how- 
ever, taken  after  the  third  injection  was  innocuous.  The 
blood  serum  obtained  by  venesection  after  the  second 
dose  of  antitoxin,  was  also  nonpoisonous.  In  addition  to 
these  experiments  Prof.  Tizzoni  found  in  the  lacerated 
finger,  amputated  on  the  same  day,  tetanus  bacilli  which 
proved  virulent  to  animals. 

Equally  favorable  was  a  case  reported  by  Dr.  G. 
Casali,  La  Riforma  Med.,  1892.  A  woman  of  twenty-two 
years  cut  her  right  foot  while  walking  barefooted.  On  the 
eighth  day  following  the  injury  she  noticed  difficulty  in 
opening  her  jaws,  and  six  days  later  the  tetanus  symptoms 
had  reached  other  muscles.  The  following  three  days 
she  received  two  injections  of  0.25  ccm.  antitoxin  daily. 
After  the  second  injection  the  symptoms  gradually  de- 
creased and  just  one  month  after  the  infection  patient  had 
perfectly  recovered.  The  fluid  discharged  from  the  origi- 
nal wound  was  examined  by  Prof.  Tizzoni  who  found,  be- 
sides other  pyogen  microbes  also  virulent  tetanus  bacilli. 

By  these  favorable  results,  obtained  with  tetanus  anti- 
toxin, quite  a  demand  was  created  for  the  same  and  Drs. 
Tizzoni  and  Cattani  had  many  occasions  to  test  its  value. 
Although  this  was  very  encouraging,  they  considered  the 
clinical  study  of  the  subject  not   yet  completed.     For  this 


32  BLOOD   SERUM  THERAPY  AND  ANTITOXINS. 

reason  they  have  so  far  not  published  their  experience 
with  reference  to  clinical  results.  It  may  however  be 
stated  that  the  mortality  in  human  tetanus,  amounting  to 
about  88  per  cent  in  the  two  largest  statistics  of  Richter 
(717  cases)  and  Forgues-Reclus  (2,072  cases)  has  been  re- 
duced to  20  per  cent  and  will  probably  be  further  reduced 
with  the  improvement  of  this  method.  A  therapy,  which 
can  cause  such  an  decrease  in  mortality,  is  certainly  won- 
derful and  ought  to  be  cheerfully  welcomed,  especially 
those  who  have  occasionally  to  witness  the  agony  brought 
on  by  this  disastrous  malady. 

The  novelty  as  well  as  the  unexpected  satisfactory  re- 
sults obtained  by  this  method,  justify  a  more  detailed  con- 
sideration of  the  following  questions,  which  are  essential 
in  securing  success  : 

1.  What  is  the  difference  of  the  immunizing  value  of  ser- 
um of  animals  of  different  species  ?  This  to  know  is  import- 
ant, because  the  immunizing  value  of  the  antitoxin  or  the 
durability  of  its  effect  may  differ  materially,  on  account  of  the 
constitution  and  physiological  properties  of  animals  of  vari- 
ous classes,  and  we  would  have  to  select  the  most  appropri- 
ate class  for  preparing  an  antitoxin  intended  for  clinical 
purposes.  The  experiments  made  in  reference  to  this, 
were  to  the  effect  that  the  immunity  is  the  more  certain 
and  lasting  the  more  homogen  (/.  e.  of  similar  species)  the 
serum  was.  For  instance,  of  three  rabbits,  inoculated  with 
serum  of  a  horse,  dog  and  rabbit  respectively,  the  one 
injected  with  the  rabbit  serum  showed  the  best  result. 
Although  being  not  fully  explained,  this  phenomenon  is  of 
great  practical  value. 
Preparation  2.     Another  important  item  is  the  preparation  of  the 

of  therapeutic  .  .  ,       ,  .  .  p      .  .  .    . 

seium.  antitoxm    and    the    estimation    of    its    immunizing   value. 

The  procedure  is  briefly  as  follows  :  The  virulent  germs 
are  cultivated  on  a  suitable  §u,bstr^te,  and  after  being  at- 


TETANUS.  33 

tenuated,  either  used  in  that  state,  or  they  are  first  filtered 
through  a  Chamberland  filter,  and  thereby  freed  from  bac- 
terial cells.  The  fluid  is  then  condensed  in  vacuo,  to 
about  one-third  of  its  original  volume,  and  in  certain  in- 
tervals, such  small  quantities  are  injected  into  a  horse,  as 
to  cause  only  a  slight  affection.  Later  on,  more  virulent 
cultures  or  their  poisonous  substrate  is  injected,  and  when 
the  animal  has  once  acquired  a  certain  tolerance  of  the 
poison,  the  dose  is  steadily  increased,  the  result  of  which 
will  be  as  Behringhas  shown,  that  the  immunizing  strength 
of  the  blood  serum  increases  accordingly.  The  injections 
are  therefore  continued  until  the  highest  possible  degree 
of  immunizing  value  has  been  reached. 

In  regard  hereto  it  is  very  important,  as  mentioned  Selection  of  the 
above,  to  know  how  susceptible  the  animal  was  before  the 
inoculation,  as  it  is  not  the  absolute  immunity  which  de- 
termines the  immunizing  value  of  the  blood  but  the  relative 
immunity,  that  is  the  difference  between  the  degree  ac- 
quired artificially  and  the  degree  natural  to  the  individual. 
The  immunizing  value  will  ^therefore  be  the  higher  the 
more  susceptible  the  animal  is  to  the  respective  infection. 
For  this  reason  one  prefers  the  horse  and  goat,  which  are 
eminently  susceptible  to  tetanus  and  diphtheria  to  other 
animals  for  the  purpose  of  obtaining  immunizing  and  ther- 
apeutic serum.  The  selection  of  the  animal,  however,  is 
not  the  only  factor  which  guarantees  the  preparation  of  a 
satisfactory  serum  ;  one  has  to  be  positive  that  the  culture 
with  which  it  is  inoculated,  is  of  such  a  nature  as  to  allow 
the  best  possible  effect. 

The  conditions  necessary  for  this  are  :     The  proper  re- ^^"^^5^^^®^°^ 
action  and  age  of  the  culture   in  addition  to  a  natural  high 
virulence  of  the  germs.     Without  observing  the  necessary 
precautions  in  this  respect  one  is  liable  to  meet  with  a  very 
annoying  occurrence,  the  decrease  of  the  virulence  of  the 


34  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

culture,  which  under  such  circumstances  may  lose  90  per 
cent  or  more  of  its  toxicity  from  one  day  to  another.  An 
alteration  of  the  toxicity  may  also  be  caused  by  exposure 
to  sunlight,  high  temperature  and  by  an  abnormal  alkalin- 
ity of  the  substrate. 

Relative  to  the  diphtheria  toxin  these  factors  are 
much  more  effective  in  tetanus  poison,  the  original  viru- 
lence of  which  can  therefore  not  be  as  easily  maintained  as 
that  of  diphtheria  toxin.  The  best  way  of  preserving  s'uch 
fluid  is  by  adding  0.5  per  cent  carbolic  acid,  or  1  per  cent 
chloroform  with  which  it  is  kept  well  sealed  in  a  cool,  dark 
place.  The  effect  the  injections  have  upon  the  animal, 
which  is  to  be  immunized,  is  visible  after  each  dose  by  in- 
creased temperature,  decrease  in  weight  and  a  certain 
change  in  the  blood,  which  is  manifested  by  the  difference 
of  coagulation.  The  separation  of  the  serum  from  the  cor- 
puscles is  retarded  and  the  former  is  not  liquid  as  usual, 
but  seems  suspended  in  a  network  of  fibrin.  During  the 
period  of  reaction  the  immunizing  value  of  the  blood  and 
consequentl}^  the  immunity  of  the  animal  is  decreased  ; 
after  the  reaction,  however,  both  increase  and  show  a 
further  increase  after  each  injection  as  long  as  the  animal 
reacts  to  the  poison.  Sometimes  the  decrease  of  the 
immunizing  value  during  a  strong  reaction  is  so  intense 
that  the  serum  becomes  toxic  (tetanus  producing)  instead 
of  immunizing.  According  to  Brieger  and  Ehrlich  the  in- 
crease of  its  value  is  again  followed  by  a  slight  decrease 
and  hereafter  remains  steady.  In  order  to  secure  a  high 
degree  of  immunity  within  the  shortest  possible  time  re- 
vaccinations  should  be  made  while  the  immunizing  value 
is  at  its  height.  The  animal  however  must  have  recovered 
from  its  temporary  illness  as  well  for  this  procedure  as  for 
the  act  of  tapping  by  which  the  therapeutic  serum  is  ob- 
tained, because  the  toxins  remain  so  long    in  the  blood  as 


TETANUS.  35 

the  individual  shows  symptoms  of  a  reaction.  Behring  and 
Knorr  advise  to  tap  an  immunized  animal  for  the  purpose 
of  procuring  therapeutic  serum  not  before  it  has  recovered 
its  normal  condition  in  regard  to  pulse,  temperature,  weight 
and  serum  separation.  By  systematic  revaccinations  of  a 
horse  with  tetanus  toxin  one  can  easily  obtain  an  immu- 
nizing value  of  1  to  ]  0,000,000  that  means  1  grm.  of  such 
serum  would  be  sufficient  to  protect  10,000,000  grm.  of 
another  animal  against  the  minimum  fatal  dose  of  this  in- 
dividual. The  experiments  of  Tizzoni  and  Cattani  have 
shown  that  the  results  in  dog  and  rabbit  differ  materially 
from  those  obtained  on  a  horse,  the  former  being  by  far 
not  as  suitable  for  furnishing  a  strong  therapeutic  serum. 
The  comparatively  highest  value  of  this  serum  was  :  In  a 
horse,  1  to  100,000,000  ;  in  a  dog,  1  to  1,000,000  ;  in  a  rab- 
bit, 1  to  1,000,000. 

We    take    occasion  at  this    time    to    emphasize  the  immunizing 

J  •  rr  ...  ,  -  serum. 

difference    between     "immunizmg    serum"     and    "  thera- Therapeutic 

serum. 

peutic  serum."  The  former  is  used  for  the  purpose  of 
protecting  an  individual  against  a  possible  infection  and 
intoxication,  or  at  least  to  mitigate  its  effect.  Therapeutic 
serum  is  applied  with  the  intention  to  neutralize  the  toxic 
effects  of  an  infection  or  intoxication  which  is  already  in 
progress.  It  is  readily  understood  that  the  serum  used  for 
this  purpose  must  be  many  times  stronger  than  immuniz- 
ing serum,  and  it  has  been  found  that  certain  proportions 
exist  between  the  two  kinds,  so  that  if  the  value  of  the  im- 
munizing serum  is  known  one  can  calculate  its  value  as 
therapeutic  serum  and  thereby  determine  the  quantity  re- 
quired for  clinical  purposes.  The  therapeutic  value  of  the 
serum  is  often  not  more  than  -^^^  or  -^-^^-^  of  the  immuniz- 
ing value  and  decreases  according  to  the  time  which  has 
elapsed  between  the  tetanus  infection  and  therapeutic  ap- 
plication. Kitasato  who  has  studied  this  question  very 
closely,  observed  the  following  facts  ; 


36  BLOOD   SERUM  THERAPY  AND  ANTITOXINS, 

Ten  mice  infected  with  tetanus  germs  showed,  forty- 
eight  hours  later,  distinct  symptoms  of  tetanus.  At  this 
time  each  of  them  received  one  ccm.  of  serum  of  an  immu- 
nized horse.  Notwithstanding  this  comparatively  large 
dose  five  mice  died  eighty  hours  after  the  infection.  The 
other  five  were  again  inoculated  with  the  equal  amount 
twenty-four  and  forty-eight  hours  after  the  first  injection. 
They  recovered  very  slowly  but  steadily,  so  that  after  sev- 
eral months  they  were  in  normal  condition.  When  instead 
of  forty-eight  hours  the  same  quantity  of  serum  was  injected 
twenty-four  hours  after  the  infection  all  animals  recovered. 
If  only  twelve  hours  had  elapsed  the  tetanus  symptoms 
were  very  light  and  could  be  checked  by  less  than  one-half 
of  the  dose.  If  the  infection  took  place  fifteen  hours  after 
instead  of  before  the  inoculation  0.001  of  the  same  serum 
was  sufficient  to  protect  a  healthy  mouse  against  the  tetanus, 
and  after  simultaneous  application  of  the  toxic  and  anti- 
toxic substance  one  ccm.  of  the  latter  prevented  the  devel- 
opment of  the  disease.  It  was  thereby  clearly  shown  that 
the  sooner  the  serum  is  applied  the  smaller  quantity  is 
required  to  have  the  same  therapeutic  effect,  and  that  the 
amount  necessary  to  neutralize  the  toxic  effect  can  be  de- 
termined according  to  the  time  which  has  elapsed  since  the 
infection. 

It  was  natural  that  after  ascertaining  these  facts  another 
question  arose  which  could  be  answered  only  by  the  most 
minute  and  delicate  investigation,  namely  :  Upon  what 
action  within  the  living  organism  is  the  curative  effect  of 
the  antitoxins  based  ?  In  regard  to  this  question  two  dif- 
ferent opinions  prevail.  According  to  Behring  and  Kita- 
sato,  the  bacterial  poisons  are  directly  neutralized  by  the 
antitoxins.  The  fact  brought  forth  as  proof  for  this  theory 
was,  that  when  the  poison  and  the  antitoxin  are  mixed  in 
certain  proportions  in  a  test  tube  the  effect  of  such  mixture 


TETANUS.  37 

injected  into  an  animal  is  usually  negative.  This  obser- 
vation is  apparently  corroborated  by  Fedoroff  who  found 
by  a  series  of  experiments,  that  if  the  tetanus  poison  and 
the  antitoxin  mixed  in  the  proportion  of  1  to  2  is  inject- 
ed into  rabbits,  the  latter  are  but  slightly  affected  ;  if,  how- 
ever, the  amount  of  tetanus  poison  contained  in  such  mix- 
ture is  injected  separately  in  a  different  place,  but  at  the 
same  time  with  the  double  quantity  of  antitoxin,  the  ani- 
mals usually  die.  He  is  therefore  inclined  to  believe  that 
the  action  of  the  poison  is  directly  balanced  by  the  anti- 
toxin. 

Contrary  hereto  are  the  results  of  Buchner's  experi- 
ments. Buchner  inoculated  twenty-three  white  mice  with  a 
certain  quantity  of  tetanus  antitoxin,  prepared  as  a  dry 
substance  from  a  beef  tea  culture,  and  also  with  an  equal 
quantity  of  antitoxin,  likewise  prepared,  to  the  effect  that 
only  three  mice  died,  and  of  the  twenty  remaining,  eleven 
were  affected  slightly  and  nine  not  at  all.  The  same 
experiment  made  on  rabbits,  which  are  much  more  suscep- 
tible to  tetanus  than  mice,  had  the  effect  that  of  twenty- 
three  animals  eight  died,  while  the  others  were  but  little 
affected. 

From  these  facts  Buchner  concluded  that  the  effect  of 
both  substances  to  each  other  is  not  a  direct  one,  but  is 
based  upon  a  certain  influence  to  the  living  cells,  which, 
being  affected  in  two  opposite  directions,  remain  neutral. 
This  theory  is  substantiated  by  several  facts.  1.  The  long 
time  before  the  antitoxin  acts.  2.  The  slow  disappearance 
of  the  tetanus  symptoms.  One  would  naturally  expect 
that  every  case  of  tetanus,  even  in  an  advanced  state, 
could  be  checked  by  antitoxin,  if  it  were  a  purely  chem- 
ical antidote.  This,  however,  is  not  the  case,  as  it  was 
observed  that  the  therapeutic  serum,  even  in  a  mild  form 
of  tetanus  does  not  relieve  the  contractions  present,  but 


38  BLOOD    SERUM  THERAPY  AND  ANTITOXINS. 

only  protects  other  parts  of  the  organism  against  the 
poison,  thereby  limiting  the  action  of  the  latter.  This 
would  explain  why  it  has  no  effect  in  advanced  stages  of 
the  disease,  and  why  a  relapse  can  occur  in  case  of  incom- 
plete immunization. 

The  following  experiment  of  Tizzoni  corroborated  this 
theory :  Some  animals  were  inoculated  with  sufficient 
quantity  of  a  virulent  tetanus  culture  to  cause  their  death 
if  not  immunized.  On  the  next  day,  after  the  first  symp- 
toms had  appeared,  they  received  antitoxin.  In  spite  of 
this,  the  local  symptoms  increased,  but  being  otherwise 
protected,  the  animals  recovered.  Thus  the  tetanus 
poison  was  limited  to  its  local  effect,  but  not  completely 
destroyed,  until  eliminated  by  the  organism.  For  these 
and  other  reasons  it  seems  that  the  living  cells  take  a  vital 
part  in  producing  immunization.  Another  point  which 
attracted  considerable  attention  was  the  question,  which 
part  of  the  organism  is  affected  by  tenanus  virus  and  anti- 
toxin. Bruchettini  found  by  his  researches  regarding  the 
distribution  of  the  poison  within  the  animal  system,  that 
from  the  place  of  infection  the  toxic  substance  is  first 
carried  away  by  the  blood,  but  that  the  larger  glands,  with 
the  exception  of  the  kidneys,  do  not  absorb  the  poison,  and 
that  part  of  it  is  eliminated  with  the  urine. 

Other  investigators,  as  Valliard,  Vincent,  Autocratow, 
and  others  showed  by  experimental  evidence  that  the 
muscles,  even  in  severe  cases,  do  not  contain  any  toxic  sub- 
stance, and  that  the  latter  causes  muscular  contractions 
merely  by  its  effect  to  the  nervous  elements.  To  the  same 
conclusions  came  C.  Brunner,  whose  clinical  studies  of  the 
subject  revealed  : 

1.  That  the  tetanus  poison  does  not  affect  directly 
muscular  tissue,  the  spasms  of  which  can  be  relieved  by 
curarizing  the  ends  of  the  motor  nerves. 


TETANUS.  39 

2.  That  no  spasms  can  be  caused  by  tetanus  poison 
in  muscles,  the  motor  nerves  of  which  have  been  sepa- 
rated from  the  centers. 

3.  That  the  muscles  of  a  certain  nerve  territory  react 
on  the  poison  only  if  the  corresponding  nerve  center  is 
irritable. 

4.  That  the  spasms  are  not  the  effect  of  irritations  of 
the  sensitive  nerves,  but  of  the  central  organs. 

With  reference  to  the  action  of  the  antitoxin,  the 
results  of  various  investigators  showed  that  the  blood  is 
the  first  but  usually  only  the  temporary  carrier  of  the 
immunizing  substance,  and  Tizzoni's  assistant.  Dr.  Cen- 
tanni,  in  his  researches  regarding  the  specific  immunization 
of  the  elements  of  tissue,  came  to  the  conclusion  that  the 
latter  and  not  the  blood  is  finally  the  one  which  is  respon- 
sible for  the  protection  of  the  organism. 

Corresponding  with  the  experimental  results  in  regard 
to  the  effect  of  toxins,  it  is  the  nerve  element  which  seems 
to  be  the  principle  absorbing  place  also  for  the  antitoxin. 
It  is,  therefore,  evident  that  if  antitoxin  is  applied  to 
neutralize  the  effect  of  the  toxin,  both  substances  will 
fight  for  their  existence  on  the  same  field,  and  that  the 
ground  held  by  the  one  cannot  be  occupied  by  the  other 
until  the  former  has  weakened  or  is  eliminated  from  the 
organism. 

The  last  problem  to  be  solved  in  the  doctrine  of  serum 
therapy,  was  regarding  the  possibility  of  inheriting  an 
acquired  immunity  against  tetanus.  Ehrlich  and  Huebener 
have  most  elaborately  investigated  this  subject  by  a  large 
series  of  experiments,  the  results  of  which  were  as  follows: 
If  the  offsprings  of  a  normal,  that  is  not  immunized  mother, 
and  an  immunized  sire  were  inoculated  with  the  minimum 
fatal  dose  of  a  tetanus  toxin,  they  invariably  died  within 
the  usual  time.     The  descendants,  however,   of  a  mother 


40  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

which  was  immune  at  the  time  of  conception,  and  a  nor- 
mal sire  was  not  affected  even  by  100-times  the  fatal 
dose.  This  clearly  shows  that  the  immunity  can  be  in- 
herited from  the  mother  but  not  from  the  sire.  The  duration 
of  such  inherited  immunity  was  found  to  be  comparatively 
short,  lasting  usually  but  two  months,  and  in  no  case 
longer  than  three  months.  These  results  are  correspond- 
ing with  those  obtained  by  Ehrlich  in  his  experiments 
with  vegetable  toxalbumins,  a  fact  which  is  considered 
important  for  the  knowledge  of  the  real  nature  of  toxic 
substances  in  general. 

In  conclusion  we  wish  to  recapitulate  the  principle 
points  to  be  considered  in  protecting  man  against  the  ef- 
fects of  tetanus  infection.  The  most  important  factor  with 
which  we  have  to  deal  is  the  gravit}-  of  the  infection  and 
the  time  since  the  latter  took  place.  The  gravity  of  an 
infection  is  measured  by  the  more  or  less  rapid  effect  upon 
the  various  groups  of  muscles. 

In  regard  to  the  treatment  of  a  person  recently  in- 
fected the  following  rules  seem  to  be  advisable  : 

1.  As  the  reaction  is  usually  proportional  to  the 
quantity  of  poison  introduced  into  the  organism,  an  early 
and  thorough  antisepsis  should  be  observed  in  dressing 
the  original  wound. 

2.  The  best  means  for  the  efficious  neutralization  of 
tetanus  germs  and  toxin  have  been  found  to  be  a  strong 
solution  of  sublimate  with  tartaric  acid  and  especially  the 
actual  cautery. 

3.  The  amputation  of  the  infected  part,  even  if  the 
latter  is  of  minor  importance,  will  hardly  afford  any  benefit, 
because  the  soluble  poison  is  rapidly  absorbed  and  car- 
ried into  circulation. 

4.  As  soon  as  the  place  of  infection  has  healed,  no 
beneficial  result   can  be   expected  from  a  local  treatment, 


TETANUS.  41 

and  no  time  should  be  lost  in  using  the  antitoxin  in  order 
to  effect  the  best  possible  immunity. 

5.  The  quantity  of  antitoxin  to  be  injected  is  deter- 
mined according  to  the  strength  of  the  latter,  the  gravity 
of  the  infection,  provided  there  are  visible  symptoms,  the 
time  since  the  infection  occurred  and  the  age  of  the  pa- 
tient. The  shorter  the  time  of  incubation  or  the  more 
serious  the  latter,  the  larger  ought  to  be  the  first  dose  of 
antitoxin. 

6.  The  number  of  injections  necessary  to  save  the 
patient  depends  also  on  such  circumstances  as  just  men- 
tioned, but  as  a  rule  one  injection  should  be  applied  every 
other  day  with  a  somev^hat  smaller  quantity  than  used  at 
first,  in  order  to  maintain  a  sufficient  immunity  and  to  pre- 
vent a  relapse,  which  occasionally  occurs  even  after  the 
immunity  had  apparently  set  in  and  which  is  due  to  a 
premature  elimination  of  the  antitoxin. 


CHAPTER  IV. 


Diphtheria. 

Far  the  greatest  interest  has  been  shown  by  the  medi- 
cal world  as  well  as  by  the  laity  to  those  discoveries  and 
experimental  results  in  blood  serum  therapy,  which  per- 
tain to  the  question  of  protection  against  diphtheria. 

Before  we  discuss  however  this  question  in  particular 
we  deem  it  proper  to  reconsider  the  theory  of  the  double 
nature  of  immunity,  which  is  fundamental  for  the  results 
obtained  by  blood  serum  therapy  and  thereby  wish  to  em- 
phasize the  difference  between  natural  and  artificial  immu- 
nity. The  theory  of  natural  immunity  is  based  upon  the 
fact  that  the  blood  and  blood  serum  posseses  certain  bac- 
tericide properties,  as  has  been  lately  proven  beyond 
doubt  by  Denys,  Kaisin,  Vaughan,  Kossel,  Hankin,  Buch- 
ner  and  others.  These  bactericide  properties  are  due  to 
soluble  substances  which  are  circulating  in  the  blood, 
called  "alexins."  The  efficacy  of  such  substances,  although 
preexisting,  can  be  raised  to  a  higher  degree  by  inoculation 
of  germs  or  their  products.  Concerning  the  nature  of  the 
alexins  we  know  very  little,  but  it  is  supposed  that  like 
the  toxic  bacterial  products  they  belong  to  the  albumins. 
In  regard  to  their  origins  the  experimental  investigations 
of  Vaughan,  Kossel  and  Buchner  have  furnished  evidence 
that  they  are  produced  within  the  leucocytes.  This,  how- 
ever, is  not  identical  with  the  theory  of  Metchnicoff,  who 
found  by  direct  microscopical  observation,  that  the  leuco- 
cytes attack  the  germs,  take  them  into  their  interior  and 
destroy  them  by  digestion.     But  such  occurrence,   called 


DIPHTHERIA.  43 

phagocytosis,  which  has  been  most  elaborately  described 
in  Senn's  *^  Principles  of  Surgery,"  cannot,  as  Metchnicoff 
believed,  bean  unquestionable  proof  for  his  theory,  because 
it  is  known  that  not  only  some  kind  of  germs  are  not  attacked 
by  the  leucocytes,  but  that  even  if  the  latter  have  been  frozen 
to  death,  the  blood  serum  still  possesses  its  former  bacteri- 
cide properties.  It  is  thereby  proved  that  phagocytosis, 
wherever  it  is  observed,  is  a  mere  secondary  feature  and 
that  the  alexins  retain  and  sometimes  even  acquire  a 
higher  degree  of  bactericide  power  by  freezing  the  serum. 
The  power  to  effectually  attack  the  germ  must  be  attributed 
to  dissolved  chemical  substances,  produced  by  the  white 
corpuscles.  The  fact  that  the  latter  frequently  take  the 
germs  into  their  interior  seems  to  be  only  an  intention  of 
nature  to  apply  such  substances  most  effectively  upon  the 
bacterial  intruder,  and  in  this  way  both  explanations  for 
the  bactericide  influence  of  the  blood  serum  stand  to 
reason. 

The  experiments  made  by  Dr.  Vaughan  to  determine 
the  nature  of  the  germicide  constituents  are  leading  to  the 
conclusion  that  the  active  substance  is  a  proteid  formed  in 
the  nuclei  of  the  cells,  and  therefore  called  nuclein.  The 
effect  of  this  substance  on  cultures  of  cholera,  anthrax 
and  staphylococcus  pyogenes  aureus  proved,  according  to 
Vaughan,  in  every  case  its  germicidal  properties.  After 
the  fact  was  established  that  such  bactericide  substances 
are  produced  in  the  white  corpuscles,  another  observation 
heretofore  misunderstood  is  now  readily  explained — the 
numerous  affluence  of  leucocytes  toward  a  place  of  infec- 
tion, and  their  abnormal  increase  in  certain  infectious  dis- 
eases. All  these  phenomena  mean  nothing  else  but  a 
defense  with  which  nature  provides  our  organism  against 
the  bacterial  offender. 

The  substances  so  produced  are  of  very  unstable  char- 


44  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

acter,  and  must  be  different  in  various  classes  of  animals  as 
well  as  in  different  individuals  of  the  same  species,  because 
they  afford,  according  to  their  antagonistic  power  against 
certain  microbes,  the  protection  which  is  known  to  be  natu- 
ral to  the  respective  class,  and,  as  mentioned  above,  called 
natural  immunity.  For  instance,  as  rats  are  naturally 
immune  against  diphtheria,  the  alexins  prepared  in  their 
leucocytes  must  differ  from  those  prepared  in  the  white 
blood  corpuscles  of  chickens,  which  are  susceptible  to  this 
disease,  but  are  immune  against  tetanus,  which  would 
seriously  affect  the  rats. 
^ntitoJins/^^  Entirely  different  from  the  cause  of  natural  immunity 

is  the  process  by  which  artificial  immunity  is  obtained.  We 
have  seen  in  our  first  chapter  that  immunity  is  the  result  of 
the  production  of  antitoxins  within  the  organism  or  their 
application  to  the  organism.  Considering  the  nature  of 
these  antitoxins  from  a  biological  standpoint,  we  have  to 
take  notice  of  their  great  resistance  against  such  influences 
as  light,  heat,  and  even  decomposition,  a  fact  which  is 
usually  not  found  in  animal  products,  and  which  rather  cor- 
roborates Buchner's  theory  that  they  are  of  bacterial  origin. 
In  accordance  herewith  is  Behring's  and  Knorr's  experience 
that  a  tetanus  culture,  after  being  attenuated  by  exposure 
to  65°  C,  sometimes  shows  directly  antitoxic  properties. 
Similar  observations  have  been  made  by  Brieger,  Kitasato, 
Klemperer,  Wassermann  and  Nencki. 

It  further  renders  the  relation  between  the  toxalbu- 
mines  and  antitoxines  at  least  possible,  if  not  probable. 
The  effect  of  both  substances  upon  each  other  has  been  dis- 
cussed in  the  chapter  on  tetanus,  and  it  may  only  be  re- 
peated that  very  likely  the  living  cell  is  instrumental  as 
well  in  producing  the  antitoxin  as  in  rendering  the  latter, 
when  introduced  as  such,  effective  against  the  toxins,  no 
matter  if  they  were  directly  inoculated  or  originated  from 
an  infection. 


No.  4.     Diphtheria  Bacilli  (Klebs-Loeffler). 

Section  of  a  diphtheria  membrane  taken  from  a  tonsil  ;  stained  in 
alkaline,  methylen  blue,  magnified  500  times.     Immersion. 

The  darker  spots  represent  thick  accumulations  of  diphtheria  bacilli 
near  the  surface  of  the  tonsil.  In  the  deeper  layers  the  bacilli  appear 
more  separated.  The  diphtheria  bacilli  are  found  exclusively  at  the 
point  of  infection,  especially  on  mucous  membranes.  They  never  appear 
in  the  blood  or  the  organs. 

The  line  where  the  dark  spots  join  a  lighter  field  represents  the 
limit  of  necrotized  tissue  (C.  Fraenkel). 


DIPHTHERIA.  45 

Besides  this,  it  should  be  remembered  that  the  anti- 
toxins are  limited  to  a  specific  effect  upon  the  correspond- 
ing poisons,  which  is  not  the  case  with  the  alexins.  The 
latter,  however,  are  acting  in  harmony  with  them  when  not 
toxic  products,  but  the  germs  themselves  are  the  object  of 
offense.  With  the  exception  of  this  occurrence  we  have 
two  well  characterized  conditions — those  produced  by  the 
alexins,  natural  immunity,  and  those  produced  by  the  anti- 
toxins, artificial  immunity.  The  principal  difference  be- 
tween these  two  groups  of  substances  is  that  the  former  are 
of  purely  animal  nature,  while  the  latter  most  probably 
originate  from  specific  bacterial  products.  In  the  immu- 
nized animal  the  antitoxic  principle  is,  however,  not  limited 
to  the  blood  serum  or  the  leucocytes,  but  is  present  as  well, 
and  occasionally  exclusively,  in  the  tissue  cells.  It  is  evi- 
dent that  under  certain  conditions  both  peculiarities,  natu- 
ral immunity,  which  would  better  be  termed  natural  toler- 
ance or  resistance,  and  artificial  immunity,  exist  and  act 
in  the  same  organism  for  the  same  purpose,  and  although 
each  may  be  raised  to  a  higher  value,  they  will  always 
retain  their  distinct  character.  Natural  resistance  can 
never  become  equal  to  specific  immunity,  nor  can  specific 
immunity  become  a  natural  resistance.  The  chief  practi- 
cal difference  between  these  two  conditions,  however,  is 
that  the  latter  is  not  transferable  to  other  individuals, 
while  specific  immunity  can  be  transferred  by  the  inocu- 
lation of  the  serum  from   individual  to  individual. 

Next  to  the  knowledge  of  these  facts,  the  admin- 
istration of  antitoxin  demands  a  certain  experience  con- 
cerning the  contagion,  intoxication  and  virulence  of  diph- 
theria bacilli  and  their  poisons.  As  generally  known,  the 
germs  which  are  now  called  diphtheria  bacilli  were  dis- 
covered by  Klebs  and  LoefHer  in  1884,  and  soon  hereafter 
the  experiments  of  Roux  and  Yersin  corroborated  the  ob- 


46  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 


1 


servations  of  the  first  named  author,  that  wherever  genuine 
diphtheria  exists  these  germs  are  present,  and  that  when 
inoculated  from  one  animal  to  another  they  reproduce  the 
disease. 

While  investigating  these  important  facts,  Hoffmann 
found  in  1887  in  many  instances  besides  the  genuine  diph- 
.  theria  bacillus  a  similar  one  which  exactly  answered  the 
description  of  the  Loeffler-Klebs  bacillus,  but  which  after 
being  isolated  and  inoculated  to  animals  proved  perfectly 
harmless.  He  therefore  called  the  latter  pseudo-diphtheria 
bacillus.  In  regard  to  the  question  whether  this  germ  is 
different  from  Loeffler's  or  only  an  attenuated  genuine 
diphtheria  bacillus  much  has  been  written,  and  the  majority 
of  bacteriologists  are  inclined  to  approve  the  opinion  of 
Roux,  Yersin  and  C.  Fraenkel,  who  believe  that  though  a 
marked  difference  is  noticeable  between  the  two  bacilli  as 
well  with  reference  to  their  virulence  as  occasionally  also 
in  their  form,  these  differences  are  not  sufficient  to  charac- 
terize the  bacilli  as  two  different  species,  but  only  as  varie- 
ties of  the  same  kind.  This  opinion  seems  the  more  plausi- 
ble if  one  considers  the  largely  fluctuating  virulence  found 
in  genuine  diphtheria  bacilli,  which  is  according  to  Roux- 
Yersin  and  Brieger-Fraenkel  the  most  important  cause  for 
the  varying  gravity  so  often  observed  in  cases  of  diphthe- 
ria. As  the  space  forbids  to  go  into  details  about  the  eti- 
ology of  this  disease  we  wish  to  mention  only  one  item 
which  is  of  importance  in  explaining  not  only  the  varying 
gravity  of  diphtheria  infection,  but  what  is  of  greater  inter- 
est to  us,  the  reason  why  in  some  cases  the  antitoxin  will 
fail  to  have  the  desired  effect.  This  occurs  when  an  infec- 
tion of  other  malign  germs  exists  simultaneously  with  that 
of  the  Loeffler  bacilli. 
Mixed  infection  It  is  an  acknowledged  fact  that  the  virulence   of  diph- 

theria   bacilli   increases  in    the    presence  of   streptococci. 


m  '  ^  ^  ^^  '^^ 


X 


No.   5.     Streptococcus  Pyogens. 

Streptococci  and  leucocytes  of  human  pus  ;  stained  in  gentian  vio- 
let, magnified  1,000  times  (Pfeiffer  and  C.  Fraenkel). 

The  streptococcus  is  a  globe  shaped  chain  forming  microbe.  It  is 
found  in  almost  every  suppurative  process,  and  is  the  cause  for  various 
pathological  conditions.  Introduced  into  the  subcutaneous  tissue  and 
the  lymphatic  system  the  streptococci  cause  the  characteristic  affection 
known  as  erysipelas  (Fehleisen).  In  diseases  of  the  inner  organs  the 
microbe  takes  a  prominent  part  on  account  of  its  virulence.  The 
streptococci  frequently  accompanies  diphtheria  bacilli,  being  an  essen- 
tial factor  as  to  the   malignancy  of  the  case. 


DIPHTHERIA.  47 

This  being  known  the  question  was  whether  the  increased 
virulence  is  due  to  a  stimulation  of  Loeifler's  bacilli  by  the 
streptococci  to  produce  more  toxin,  or  if  the  organism  is 
rendered  more  susceptible  to  diphtheria  by  the  other 
germs.  In  order  to  answer  this  problem  Funk,  who  has 
devoted  considerable  interest  to  the  study  of  such  mixed 
infection,  made  two  series  of  experiments  in  the  Berlin  in- 
stitute of  infectious  diseases. 

First  he  tried  to  ascertain  what  effect  a  certain  quan- 
tity of  streptococci  would  have  when  inoculated  to  a  guinea 
pig  with  diphtheria  toxin  of  a  tested  strength.  The  effect 
was  measured  by  a  quantity  of  antitoxin  sufficient  to  neu- 
tralize the  toxin  and  the  experiment  led  to  the  result  that  the 
animals  did  not  show  an  increased  susceptibility  for  the  tox- 
in. Of  more  positive  effect,  however,  were  the  experiments 
made  with  streptococci  in  combination  with  living  diphthe- 
ria cultures  instead  of  the  ready  toxin.  The  antitoxin  used  in 
the  animals,  inoculated  with  this  mixed  material  was  in  no 
case  sufficient  to  protect  the  animals  against  infection,  which 
resulted  in  death  in  about  50  per  cent,  while  the  animals 
which  received  an  equal  amount  of  diphtheria  cultures  and 
antitoxin  but  no  streptococci  all  remained  well.  Thereby 
the  interesting  fact  was  established  that  the  streptococci 
have  a  stimulating  effect  upon  the  productive  ability  of 
diphtheria  bacilli,  which,  however,  can  occasionally  be 
compensated  by  an  accordingly  increased  dose  of  thera- 
peutic serum.  We  shall  return  to  these  important  facts 
when  speaking  of  the  way  in  which  a  neutralization  of  the 
diphtheria  poison  is  effected  by  the  antoxin.  Being  fa- 
miliar with  the  phenomenon  that  conditions  as  mentioned 
above  result  in  a  different  intensity  of  diphtheria  infection, 
a  practical  use  of  this  fact  was  first  made  by  Behring  and 
Wernicke,  and  later  by  H.  Aronson  who  endeavored  to 
ascertain    the  degree    of  virulence    of  different  cases  and 


48  BLOOD    SERUM  THERAPY  AND  ANTITOXINS. 

succeeded  in  determining  how  to  obtain  pure  cultures  of  a 
standard  efficacy.  Aronson  further  found,  that  the  cultures 
from  cases  of  different  severity  were  of  so  much  varying 
virulence  that  in  one  instance  1  to  2  grms.,  in  others  but 
0.05  grm.,  of  a  culture  was  necessary  to  cause  death  to  a 
guinea  pig.  These  results  of  experimental  studies  explain 
very  clearly  the  variety  of  single  cases  as  well  as  of  whole 
diphtheria  epidemics. 

A  material  progressin  the  researches  regarding  the  viru- 
lence of  diphtheria  bacilli  was  made  by  Roux  and  Yersin's 
observations  that  the  virulence  can  be  raised  by  cultivating 
the  bacilli  either  on  a  suitable  nutrient  substrate  together 
with  streptococci,  or  by  passing  the  germs  through  a  series 
of  animal  organisms,  a  method  by  which  under  favorable 
conditions  as  to  the  individual  susceptibility,  etc.,  a  viru- 
lence 100  times  stronger  than  that  of  the  original  culture 
can  be  secured.  The  contrary  effect,  an  attenuation  of  an 
originally  virulent  culture  can  be  obtained  by  passing  the 
germs  through  animals  with  little  or  no  susceptibility  for 
diphtheria.  The  so  attenuated  germs  maintain  their  re- 
duced virulence  until  they  are  placed  again  upon  a  more 
favorable  soil,  on  which  they  may  regain  their  full  viru- 
lence. These  facts  are  of  eminent  importance  for  the 
preparation  of  diphtheria  therapeutic  serum,  the  principles 
of  which  ma}'  be  stated  in  the  following  notes  : 

We  have  mentioned  that  the  quantity  of  antitoxin 
obtainable  depends  mainly  upon  the  susceptibility  of  the 
animal.  Therefore  one  'would  have  to  select  a  species 
which  warrants  a  good  quality  of  serum  by  a  high  suscepti- 
bility for  diphtheria  poison.  The  most  useful  objects  for 
the  purpose,  especially  in  laboratory  work  are  goats,  which 
by  Ehrlich  and  Wassermann  are  considered  preferable  to 
other  animals,  not  only  on  account  of  the  high  susceptibil- 
ity they  possess,  but  also  because  they  show  a  great  resist- 


DIPHTHERIA.  49 

ance  when  sick  from  inoculations,  and  further  afford  an 
opportunity  to  use  their  milk  which  possesses  considerable 
antitoxic  value.  Thus  we  have  in  these  animals  all  the 
advantages  combined  which  enable  us  to  secure  in  regard 
to  quality  as  to  quantity  the  most  efficient  material. 

The  method  of  immunizing  consists  in  creating  a  tol- 
erance of  the  diphtheria  poison  by  systematic  inoculation 
with  at  first  attenuated  diphtheria  cultures,  later  on  more 
virulent  cultures,  and  finally  by  direct  application  of  the 
toxin.  As  the  tolerance  or  resistance  against  the  poison 
increases  so  does  the  immunizing  power  of  the  blood  by 
production  of  antitoxin. 

The  toxin  used  for  the  inoculation  is  obtained  in  the 
following  way:  From  a  colony  of  diphtheria  germs  culti- 
vated on  a  Petri  dish  a  quantity  is  added  to  a  quart  of  beef 
tea  in  a  large  flask.  The  latter  remains  three  or  four  weeks 
in  an  incubator,  during  which  time  the  germs  saturate  the 
fluid  with  their  toxic  products.  By  adding  ^^  per 
cent  carbolic  acid  or  yz  per  cent  trikresol  the  bacilli  are 
then  precipitated  to  the  bottom  of  the  flask  and  the  liquid 
now  contains  the  toxin  in  solution.  From  this  solu- 
tion the  toxin  can  either  be  prepared  as  a  dry  substance, 
which  is,  however,  very  expensive,  or  it  can  be  used  while 
dissolved  in  the  fluid  after  filtration  through  a  Chamber- 
land  filter.  The  toxin  is  now  ready  for  injection,  the  ef- 
fect of  which  is  local  swelling,  fever  and  the  production  of 
antitoxin. 

The  latter  is  found  in  the  blood  after  each  injection  valuation  of 
and  increases  in  quantity  with  the  number  of  injections. 
For  the  estimation  of  its  efficacy  it  is  necessary  to  tap  the 
animal  from  time  to  time  for  a  small  quantity  of  blood 
with  which  experiments  are  made  on  others.  The  mode 
of  valuation  is  based  upon  the  observation  of  Behring  and 
Kitasato  that    toxin   and    antitoxin    neutralize    each   other 


50  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

when  mixed  in  certain  proportions  in  a  test  tube.  Differ- 
ent from  this  is  the  method  previously  used,  by  separate  in- 
jections of  toxin  and  antitoxin,  which  however  does  not 
give  as  exact  results  on  account  of  the  varying  absorbing 
ability  of  the  animal.  With  administration  of  the  former 
method  an  examination  of  the  blood  samples  is  made  ac- 
cording to  Ehrlich  in  the  following  way:  Of  a  toxin  of  a 
standard  strength  0.1  grm.  representing  the  minimum  fatal 
dose,  for  a  guinea  pig  weighing  300  grms.,  1  grm.  is 
mixed  with  a  number  of  different  quantities  of  the  blood 
to  be  examined,  for  instance,  0.4  grm.,  0.3  grm.,  0.2 
grm.,  0.1  grm.,  and  with  these  four  mixtures  as  many 
guinea  pigs  are  inoculated.  The  result  will  be  that  cor- 
responding with  the  amount  of  serum  a  different  local  and 
general  effect  takes  place. 

The  animal  which  received  the  largest  dose  will  show 
no  reaction.  The  one  that  received  0.3  grm.  of  serum 
may  suffer  from  an  acute  local  inflammation,  followed  by 
necrosis,  but  otherwise  be  not  much  affected.  The  third 
will  probably  become  quite  sick,  and  the  one  which  got  the 
smallest  dose  of  antitoxin  will  die  within  forty-eight 
hours  from  the  effect  of  the  same  quantity  of  toxin  as  the 
other  received.  In  this  way  we  can  determine  which 
amount  of  blood  of  the  immunized  animal  is  sufficient  to 
neutralize  a  certain  quantity  of  toxin,  and  so  the  value  of 
the  serum  can  be  established. 

The  absolute  estimation,  or  an  exact  figure  of  its  value, 
can  be  given,  if  one  has  a  material  of  a  standard  strength 
with  which  to  compare  others.  Behring  and  Ehrlich  there- 
fore prepared  a  serum  of  which  0.1  grm.  is  sufficient  to 
neutralize  1  grm.  toxin  (ten  times  the  fatal  dose  for  a 
guinea  pig  of  300  grms.),  and  this  they  called  ^'Normal 
Therapeutic  Serum."  Of  such  normal  therapeutic  serum 
1  ccm.  is  termed  the  equivalent  for  one    immunizing  unit. 


irf^^^^?fe:> 


No.  6.     Diphtheria  Bacilli. 

Culture  on  agar-agar,  twenty-four  hours  old  ;  stained  in  alkaline 
methylen  blue,  magnified  1,000  times. 

Diphtheria  bacilli  is  best  cultivated  on  a  substrate  of  agar,  or  blood 
serum.  Macroscopically  they  appear  as  a  shiny,  granulated  mass  ; 
microscopically  they  present  short,  irregular  bacilli,  frequently  showing 
on  one  end  the  shape  of  an  olive. 


DIPHTHERIA.  51 

Accordingly  a  serum,  of  which  0.01  grm.  is  sufficient  to 
neutralize  ten  times  the  fatal  dose,  would  possess  ten 
immunizing  units  or  would  be  a  ten  fold  normal  serum; 
if  0.001  grm.  has  the  same  effect,  1  grm.  of  the  serum 
would  have  100  immunizing  units,  etc.  Has  the  value  of 
the  serum  once  been  established,  the  animal  is  tapped 
for  a  Istrger  quantity  of  blood,  which,  being  received  in 
sterilized  vessels,  is  placed  on  ice  until  coagulation  has 
been  completed  and  the  serum  been  separated  as  a  clear, 
yellowish  fluid.  This  fluid  with  0.5  per  cent  carbolic 
acid  now  presents  the  material  to  be  used  for  therapeutic 
purposes. 

It  is  of  special  importance  to  remember,  that  in  pre- 
paring the  serum  it  is  not  our  aim  to  accomplish  a  high  de- 
gree of  immunity  in  the  animal,  but  to  avoid  this  as  much 
as  possible  because  the  longer  the  animal  show  its  sus- 
ceptibility, the  better  will  be  the  serum  it  furnishes  in  rela- 
tion to  the  increased  production  of  antitoxin  in  its  system. 
As  the  valuation  of  the  serum  is  of  vital  importance  for  the 
practical  administration,  it  seems  justified  to  consider  this 
subject  somewhat  more  explicitly. 

Although  the  principles  of  the  above-mentioned  method 
have  been  recognized  as  most  practicable  when  the  experi- 
ments with  antitoxins  were  still  in  their  earliest  stage,  spe- 
cial attention  was  given  the  subject  by  Behring  and  Knorr 
in  their  publication  of  '' Infection  and  Disinfection."  Later 
the  methods  of  valuation  were  repeatedly  modified  according 
to  Behring- Wernicke's  and  Behring-Boer's  investigations. 
After  discovery  of  diphtheria  antitoxin  the  method  of  mix- 
ing toxin  with  antitoxin  was  at  first  the  one  usually  em- 
ployed; for  several  reasons  however  it  was  temporarily 
abandoned;  first,  because  the  preparation  of  a  toxin 
strong  enough  to  effect  death  in  very  small  quantity  met  at 
that  time  with  great    difficulties,    and    second,   because  it 


52  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

was  believed  that  the  degree  of  immunity  obtained,  would 
directly  indicate  the  value  of  the  antitoxin,  produced  in 
the  animal,  so  that  the  strength  of  the  serum  could  simply 
be  determined  by  the  amount  of  the  inoculated  poison.  If 
this  were  correct  it  would  be  unnecessary  to  enter  into  the 
complicated  examination  of  blood  samples,  by  which  toxin, 
antitoxin  and  animals  are  consumed.  But  the  investiga- 
tions of  Behring  and  Wernicke  concerning  the  relation  of 
immunity  to  therapeutic  results  in  diphtheria  showed  that 
a  staple  relation  between  the  acquired  immunity  and  the 
quantity  of  antitoxin  does  not  exist.  On  the  contrary,  as 
was  mentioned  in  the  chapter  of  tetanus,  Behring  found 
that  occasionally  an  animal  may  possess  a  large  quantity  of 
antitoxin  and  at  the  same  time  a  higher  susceptibility  than 
before. 

In  face  of  these  facts  the  method,  based  upon  the  idea 
that  a  certain  relation  between  immunity  and  antitoxin  ex- 
isted, had  to  be  abandoned.  By  later  investigations, 
Behring  and  Boer  attempted  to  solve  another  question, 
namely,  how  the  effect  of  antitoxin  in  an  animal,  inoculated 
with  virulent  cultures,  compares  with  the  effect  of  an  equal 
dose  of  antitoxin  in  an  animal  inoculated  with  the  ready 
toxin. 

Previous  to  this  the  efficacy  of  the  normal  serum  was 
estimated  by  its  power  to  save  an  animal  after  inocula 
tion  with  ten  times  the  fatal  dose  of  a  living  culture, 
which  was  considered  an  analogical  procedure  to  the  in- 
fection of  patients.  On  account  of  this  analogy,  it  was 
believed  that,  after  the  difference  of  the  effect  in  animals 
had  been  established,  and  so  the  necessary  dose  of  antitox- 
in as  a  prophylactic  and  as  a  curative  had  been  deter- 
mined, the  result  would  also  apply  to  man.  This  conclu- 
sion, however,  was  found  to  be  incorrect,  and  the  result  of 
many  observations  on  persons  showed,  that   the  amount  of 


DIPHTHERIA.  53 

antitoxin  required  to  cure  diphtheria  infected  patients, 
can  only  be  estimated,  aside  from  due  consideration  of  the 
different  weight,  by  comparative  calculation  of  the  quan- 
tity of  ready  toxin  and  not  of  the  virulent  cultures  given  to 
the  animal. 

How  important  this  discovery  was,  is  readily  under- 
stood, if  one  compares  the  quantity  of  antitoxin  required 
to  save  an  animal  after  infection  with  living  cultures,  and 
that  necessary  to  save  it  after  inoculation  with  the  ready 
toxin.  A  large  series  of  experiments  made  in  this  respect, 
led  to  the  result,  that  the  dose  of  antitoxin  necessary  to 
neutralize  ten  times  the  minimum  fatal  dose  of  toxin,  is 
fifty  times  larger  than  that  for  the  equal  amount  of  virulent 
cultures.  In  accordance  herewith,  Behring  and  Boer 
stated,  that 

1.  1  ccm.  of  their  "Normal  serum,"  injected  sepa- 
rately from  the  poison,  saves  5,000  grms.  of  animal  from 
death,  after  inoculation  with  the  tenfold  fatal  dose  of  a 
two  days  old  diphtheria  culture. 

2.  1  ccm.  of  normal  serum,  injected  separately  from 
the  poison,  saves  100  grms.  of  animal  from  death,  after 
inoculation  with  the  tenfold  fatal  dose  of  toxin. 

3.  0.1  ccm.  of  normal  serum  neutralizes  in  a  test 
tube,  at  least  ten  times  the  fatal  dose  of  toxin  for  a  guin- 
ea pig  of  300-400  grms.  The  accuracy  of  these  figures 
necessitate  of  course,  a  steady  toxicity  of  the  poison 
and,  knowing  that  the  latter  varies  considerably,  we 
would  have  to  establish,  what  is  the  fatal  dose  or 
what  is  the  strength  of  the  toxin,  to  which  the  above 
figures  apply.  As  the  individual  disposition  represents 
a  strong  factor  in  the  estimation  of  the  toxicity,  we  can 
only  calculate  the  fatal  dose  of  taking  the  average  from 
experiments  on  a  larger  number  of  animals.  If,  for  in- 
stance, ten    guinea  pigs,  weighing   between   200   and    400 


54  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

grms.,  or  together  3,000  grms.  were  inoculate  with  1 
grm.  diphtheria  toxin,  and  three  of  them  die,  while  the 
rest  after  more  or  less  serious  affections,  recover,  we  would 
consider  1  grm.  the  minimum  fatal  dose  for  8,000  grms. 
living  weight,  or  0.1  grm.  for  one  animal. 

With  reference  to  a  diphtheria  culture  in  beef  tea  the 
fatal  dose  is  considerable  smaller,  about  0.005  ccm.,  pro- 
vided the  culture  was  well  developed  by  being  kept  in  an 
incubator  for  two  or  three  days. 

The  quantity  of  toxin  which  represents  the  fatal  dose 
having  been  established,  a  complete  valuation  of  a  serum 
would  require  four  series  of  experiments,  intending  to  de- 
termine : 

1.  The  immunizing  value  after  an  infection  (inocula- 
tion with  cultures). 

2.  The  curative  value  after  an  infection. 

3.  The  immunizing  value  after  an  intoxication. 

4.  The  curative  value  after  an  intoxication. 

The  results  of  such  experiments  are  the  figures  men- 
tioned above  under  1  and  2.  The  most  essential  point  of 
these  discoveries  is  the  fact  that 

1.  The  administration  of  antitoxin  is  perfectly  harm- 
less. 

2.  It  positively  possesses  prophylactic  and  curative 
properties,  if  applied  in  the  proper  quantity. 

Of  what  effect  these  properties  of  the  serum  would  be 
on  men  had  of  course  to  be  investigated  on  patients.  The 
numerous  observations  in  German  and  French  hospitals  by 
noted  medical  authorities  gave  abundant  evidence  to  cor- 
robate  the  results,  previously  obtained  on  animals.  In  all 
of  these  the  correct  estimation  of  the  necessary  dose  was 
instrumental  not  only  in  getting  a  definite  judgment  of  the 
merits  of  this  treatment,  but  also  in  convincing  those  mem- 
bers of  the  profession,  whose  doubts  and  prejudice  against 


»     ''Ka^%  ^■ 


.-'v 


',  f' 


No. 


Diphtheria  Bacilli, 


Culture  on  blood  serum,  prepared  as  No.  6,  magnified  1,000  times. 

The  short  form  presented  in  this  specimen  is  due  to  their  rapid 
multiplication.  Some  of  the  germs  are  distinguished  by  a  club  formed 
shape,   which  is   considered  characteristic  to  this  species. 


DIPHTHERIA.  55 

this  therapy  rendered  them  unable  to  comment  upon  the  . 
value  of  the  so  minutely  conducted  experiments.  Thou- 
sands of  animals  have  been  sacrificed  in  order  to  arrive  at 
certain  conclusions  about  the  utility  of  this  therapy  and 
having  passed  the  experimental  stage  it  was  given  a  trial 
even  by  the  most  conservative  scientists.  The  clinical  ex- 
perience of  many  observers  extending  over  several  years 
not  only  unanimously  agrees  upon  the  high  practical  value 
of  the  new  doctrine  but  is  in  itself  a  contribution  to  our 
knowledge  as  to  the  dosage  and  the  therapeutic  limits  of 
blood  serum   therapy. 

We  now  will  direct  our  attention  to  the  establishment  Dosage  of 

antitoxin. 

of  the  correct  dosage  and  to  certain  conditions  to  be  con- 
sidered in  the  administration  of  this  therapy.  As  explained 
above,  a  much  larger  dose  of  therapeutic  serum  is  necessary 
to  neutralize  the  effect  of  an  intoxication  than  that  of  an 
infection.  This  appears  quite  natural  as  the  serum  acts  in 
both  cases  against  the  toxin  present,  which  after  an  infec- 
tion is  but  originating  and  therefore  easier  neutralized  than 
the  corresponding  dose  of  ready  toxine.  For  the  same 
reason  it  is  evident  that  it  requires  a  larger  dose  of  anti- 
toxin to  cure  a  case  of  several  days'  standing  than  to  check 
an  infection  in  its  incipiency,  because  in  the  former  the 
bacilli  have  produced  considerable  more  toxin.  How 
much  more  antitoxin  is  required  in  an  advanced  case  of 
diphtheria  cannot  be  exactly  stated  and  depends  mainly  on 
the  gravity  of  the  case,  but  it  is  certain  that  the  effect  is 
proportional  to  the  dose,  as  noticed  on  animals,  which  re- 
ceived more  antitoxin  than  necessary  to  neutralize  a  pre- 
vious diphtheria  infection.  For  instance,  a  guinea  pig,  if 
infected  with  ten  times  its  fatal  dose,  would  be  saved  by  a 
quantity  of  antitoxin  in  the  proportion  of  1  to  5,000  of  its 
weight,  but  not  without  showing  signs  of  severe  illness. 
The  latter  would   be  materially  lessened  by  a  dose  of  the 


56  BLOOD   SERUM  THERAPY  AND  ANTITOXINS. 

proportion  of  1  to  2,000  of  animal  weight  and  may  not  set 
in  at  all  after  a  dose  of  1  to  500. 

The  experiments  made  on  children  with  blood  serum 
showed  that  at  least  500  immunizing  units  are  necessary  to 
effect  a  cure  even  in  light  cases  of  diphtheria,  that  is  50 
ccm.  if  the  material  is  a  tenfold  normal 'serum,  or  10  ccm. 
if  it  is  fifty  times  as  effective.  In  more  serious  or  advanced 
cases  1,000  to  1,500  immunizing  units  would  be  required  to 
save  the  child  and  as  there  are  many  factors  which  may  be 
apt  to  aggravate  a  case,  it  will  always  be  a  matter  of  per- 
sonal judgment  how  to  class  the  respective  case. 
It  forms  An  item  also  worthy  of  consideration  is   the  fact  that 

several  cases  bemg  apparently  under  the  same  condition, 
take  an  entirely  different  course.  In  the  same  family,  for 
instance,  one  child  may  present  only  the  common  picture  of 
angina  and  recover  in  a  few  days.  Another  child  may  have 
laryngeal  membranes  which  necessitate  tracheotomy  or  in- 
tubation. A  third  one  may  show  from  the  beginning  a 
serious  affection  of  the  tonsils,  throat  and  nose  with  ob- 
structions by  thick  membranes  and  suppuration  from  the 
effect  of  which  it  could  not  be  saved  even  by  early  tra- 
cheotomy. These  cases,  though  having  originated  from  the 
same  cause,  the  infection  with  diphtheria  germs,  would  re- 
quire very  different  doses  of  antitoxin.  In  order  to  deter- 
mine the  necessary  dose  it  must  be  remembered  that  the 
latter  should  be  proportional  to  the  toxin  present  which  as 
is  well  known  increases  rapidly  from  day  to  day  after  an 
infection. 

A  curative  effect  can  therefore  with  certainty  be  ex- 
pected only  if  the  organism  has  not  too  long  been  preoccu- 
pied by  the  toxin,  that  is,  in  an  early  stage,  or  in  a  milder 
form  of  the  disease.  When  the  diphtheric  process  already 
affects  the  bronchi  and  lungs, so  that  even  tracheotomy  is  of 
no  avail,   the  application   of  serum  will  hardly  do  better. 


DIPHTHERIA,  57 

The  prognosis  is  always  doubtful  if  the  toxin  has  circulated 
for  a  longer  period,  say  three  to  four  days,  because  their 
paralyzing  effect  upon  the  nerves  and  ganglia  of  the  heart 
can  no  more  be  eliminated.  The  most  important  factors 
of  aggravation  of  a  case  is  the  complication  by  a  simulta- 
neous infection  with  other  germs,  especially  streptococci, 
and  it  is  in  such  cases  of  mixed  infection  that  we  find  the 
most  virulent  diphtheria  bacilli. 

In  order  to  intelligently  comprehend  the  character  of  ^^xedfnfectio 
these  cases  and  to  express  an  opinion  as  to  the  utility  of 
antitoxin  it  seems  necessary  first,  to  understand  the  po- 
sition and  effect  of  the  nondiphtheric  microbes.  According 
to  the  observations  of  Barbier,  Schrader,  Roux  and  Yersin 
the  infection  with  diphtheria  bacilli  present  a  more  serious 
character  when  mixed  with  other  germs,  especially  strep- 
tococci. The  latter  penetrate  the  mucous  membrane  of 
the  tonsils  and  are  carried  to  the  lymphatic  glands  which, 
affected  by  the  bacterial  products  become  oedematous  and 
finally  turn  into  suppuration. 

Aside  from  these  complications  the  diphtheria  bacilli 
become  more  virulent  in  the  presence  of  streptococci. 
Starting  from  this  fact  Funk  tried  to  ascertain  whether  the 
diphtheria  bacilli  really  produce  a  stronger  toxin  or  if  the 
organism  is  rendered  more  susceptible  for  the  poison  by  said 
complications.  He  inoculated  twenty-two  guinea  pigs  with 
a  certain  multiple  of  the  fatal  dose  of  diphtheria  culture,  to 
which  in  eleven  animals  a  culture  of  streptococci  was  added. 
Twenty-four  hours  before  the  inoculation  all  animals  had  re- 
ceived a  sufficient  quantity  of  antitoxin  to  neutralize  the  diph- 
theria infection.  The  result  was,  that  the  animals  with  mixed 
infection  died,  while  the  others  recovered.  If  instead  of  a 
living  culture  the  ready  toxin  was  applied  the  streptococci 
had  no  aggravating  effect.  It  was  proved  by  these  experi- 
ments that  streptococci  render  the  diphtheria  bacilli  more 


58  BLOOD    SERUM  THERAPY  AND  ANTITOXINS. 

virulent.  Knowing  this  fact,  one  should  expect  that  an  ac- 
cordingly larger  dose  of  antitoxin  would  balance  the 
increased  toxicity,  and  to  a  certain  extent  this  is  really  the 
case. 

But  as  mentioned  above  the  streptococci  produce  other 
symptoms  which  are  apt  to  decrease  the  vitality  and  resist- 
ance of  the  patient.  Against  these  complications  nothing 
can  be  expected  from  the  antitoxin.  It  may,  however,  be 
possible  to  check  a  mixed  infection  by  application  of  two 
different  antitoxins,  one  acting  against  the  diphtheria  tox- 
in, the  other  intended  to  neutralize  the  effect  of  the  pro- 
ducts of  streptococci.  Being  at  present  engaged  in  these 
experiments  we  hope  to  come  soon  to  a  conclusion  as  to 
their  practical  value. 

From  the  facts  above  mentioned  we  arrive  at  the  fol- 
lowing conclusions  in  regard  to  serum  therapy  :  1.  Every 
case  should  be  treated  in  its  incipiency  locally  with  such 
antiseptics  as  seem  fit  to  suppress  a  mixed  infection. 
2.  The  antitoxin  will  be  of  so  much  better  effect  even  in 
cases  of  mixed  infection  the  sooner  a  proper  dose  of  anti- 
toxin is  applied. 

The  local  treatment  with  antiseptics  is  not  to  be  neg- 
lected for  another  reason.  The  germs  developing  on  the 
tonsils  extend  their  effect  not  only  to  the  case  in  question, 
but  may  be  instrumental  for  the  infection  of  others.  Even 
convalescents  carry  the  virulent  germs  sometimes  for  along 
period  after  all  local  symptoms  have  disappeared.  In  some 
instances  diphtheria  bacilli  were  found  as  long  as  eight 
weeks  after  recovery,  a  fact  which  deserves  special  atten- 
tion in  reference  to  school  hygiene.  In  other  cases  an 
apparent  recovery  is  followed  by  secondary  affections  of 
the  kidneys,  peripheric  nerves,  etc.,  also  due  to  diphtheria 
intoxication,  and  in  face  of  such  complications  it  may  seem 
that  the  virtues  of  antitoxin  were  but  very  limited.     Con- 


DIPHTHERIA.  59 

sidering,  however,  that  if  the  production  of  the  toxin  is 
neutralized  in  an  early  stage,  such  serious  results  are  not 
very  liable  to  occur,  we  come  again  to  the  conclusion  that 
everything  depends  upon  the  early  application  of  the  anti- 
toxin as  well  as  an  appropriate  local  treatment.  In  regard 
to  secondary  affections  the  experiments  of  P.  Meyer  have 
shown  that  diphtheria  paralysis  is  due  to  degeneration  of 
nerve  fibers,  caused  by  the  toxin.  Is  the  latter  effectually 
checked  by  antitoxin,  the  patient  will  have  a  good  chance 
to  escape  such  complication. 

Knowing  the  various  factors  which  are  of  importance 
regarding  the  efficacy  of  serum  therapy  we  will  be  able  to 
consider  more  intelligently  the  results  so  far  obtained. 
According  to  Roux  and  Martin's  observations  in  Paris  the 
effect  of  antitoxin  was  most  favorable  in  cases  in  which 
tracheotomy  was  performed.  The  mortality  of  these  cases 
averaged  during  the  last  five  years  about  85  per  cent,  and 
was  reduced  by  serum  therapy  to  less  than  47  per  cent. 
At  the  same  time  many  others  which  previously  would  have 
had  to  be  operated  were  cured  by  this  new  therapeutic 
agent  without  surgical  aid. 

Even  better  results  than  these  are  shown  by  statistics  statistic. 
from  the  hospitals  of  Berlin.  In  most  of  the  cases  the  diag- 
nosis was  made,  based  on  bacteriological  examinations  and 
such  cases  which  started  with  diphtheria  symptoms,  but 
later  proved  to  be  scarlet,  measles,  etc.,  were  not  considered 
in  the  following  figures  :  Of  233  cases  reported  by  Kossel 
179  recovered,  which  is  equal  to  77  per  cent.  In  these  are 
included  72  children  with  tracheotomy,  of  whom  41  recov- 
ered, representing  57  per  cent  against  but  25  per  cent  in 
former  years. 

To  show  how  the  results  of  serum  therapy  differs  ac- 
cording to  the  age  of  the  patient  and  stage  of  the  disease, 
we  add    the  inclosed  tabulae.      From   tabula  1   it  is  evident 


60  BLOOD    SERUM  THERAPY  AND  ANTITOXINS. 

that  the  high  percentage  of  recoveries  after  tracheotomy 
is  chiefly  due  to  the  good  results  obtained  in  children 
under  two  and  five  years,  while  without  operation  the 
older  ones  show  better  results  than  the  others. 

More  interesting  than  this  is  tabula  2,  which  repre- 
sents the  same  233  cases  divided  into  groups  according  to 
the  day  when  the  treatment  was  commenced.  The  re- 
sults obtained  in  the  first  two  days,  speak  for  themselves, 
and  need  no  further  comment.  The  statement  relating  to 
the  advanced  cases  is  all  the  more  remarkable  as  the 
determination  when  a  case  began,  depended  mainly  upon 
the  dates  given  by  relatives,  and  for  this  reason  it  seems 
just  to  presume,  that  in  many  cases  the  disease  had 
started  sooner  than  observed  by  them. 

The  results  of  serum  therapy,  as  given  in  tabula  2, 
are  in  harmony  with  the  effect  seen  in  experiments. 
The  longer  the  toxin  had  circulated  in  the  organism, 
and  the  more  complicated  the  case  was,  either  on  ac- 
count of  obstructions  in  the  respiratory  organs  or  on  ac- 
count of  the  virulence  of  the  infection,  the  less  effect 
had  the  antitoxin.  The  antitoxin  used  in  the  cases 
reported  in  the  above  tabulae,  was  fifty  to  sixty  times  as 
strong  as  Behring's  normal  serum,  so  that  1  ccm.  con- 
tained fifty  to  sixty  immunizing  units. 
Effect  to  local  The  dosc  administered,  varied  according  to  the  case 

symptoms.  betwccu  160  to  800  uuits.  The  injections  were  usually 
made  on  the  lateral  part  of  the  chest.  The  fluid  was 
absorbed  within  one  to  two  hours,  producing  more  or 
less  pain,  which  gradually  disappeared  in  course  of 
twenty-four  hours.  An  immediate  reaction  regarding  the 
temperature  has  in  no  case  been  observed,  but  in  many 
instances  a  skin  eruption  like  urticaria  appeared,  several 
days  and  as  late  as  two  weeks  after  the  injection.  This 
has    more    frequently  been    noticed    when    the    serum    of 


RESULT   OF   ANTITOXIN    IN    DIPTHERIA. 

Tabula   I. 


ii 

1 

si 

With  Tracneotomy. 

•s 

i 

S 

5Z5 

a' 

0-1 
1-2 
2-3 
3  4 
4-5 
5  6 
6-7 
7-8 
8-9 
9  10 
10-11 

1 

16 
35 
40 
34 
23 
16 
21 
19 
12 
6 
7 

'"s" 

27 
30 
24 
19 
10 
16 
17 
12 

6 

7 

1 
8 
8 
10 
10 
4 
6 
5 
2 
0 
0 
0 

50 

77 

75 

70 

83 

62.5 

76 

89.5 
100 
100 
100 

1 

8 

14 
17 
11 
5 
8 
4 
3 
1 

''*2   ' 
8 

13 
8 
2 
4 
0 
3 
1 

1 
6 
6 
4 
3 
3 
4 
4 
0 

0 

25 

57 

77 

73 

40 

50 

0 

100 

100 

11-12 

12-13 

13-14 

2 
1 

2 

1 

100 
100 

over  14 

233 

179 

54 

72 

41 

31 

Tabula  II  * 


st- 

o 

a 

a& 

1^ 

•SI 

>> 
1 

d 

II 

IS 

IS 

q 

Z 

tf 

Q 

0^ 

I. 

7 

7 

0 

100 

II. 

71     (  9) 

69 

(  7) 

3     (  2) 

97 

III. 

30     (  7) 

26 

(  6) 

4(1) 

87 

IV. 

39     (14) 

30 

(10) 

9     (  4) 

77 

V. 

25     (11  ) 

15 

(  5) 

10     (  6) 

61) 

VI. 

17     (  7) 

9 

(  2) 

8(5) 

47 

VII. 

41     (23  ) 

21 

(10) 

20     (13) 

51 

VIII. 

3(1) 
233  (72  ) 

2 
179 

(  1  ) 
(41  ) 

1 



54     (31  ) 

77 

*The  figures  in  parentheses  refer  to  cases  in  which  tracheotomy  was 
performed. 


DIPHTHERIA.  61 

sheep  or  dogs,  than  if  that  of  goats  or  horses  had  been 
used.  It  proved  to  be  in  all  cases,  a  very  harmless  oc- 
currence, which  had  no  relation  to  the  efficacy  of  the 
serum. 

With  reference  to  the  local  symptoms,  a  direct 
change  after  the  injection  of  serum  does  not  take  place. 
The  white  membranes  were  frequentl}^  found  larger  on 
the  next  day,  than  they  were  before,  but  notwithstand- 
ing this  apparent  progress,  the  disease  had  not  advanced. 
Especially  the  larynx,  became  in  no  case  involved  after 
the  application  of  antitoxin,  if  it  had  not  been  affected 
previously,  and  even  many  of  those  in  which  laryngeal 
symptoms  were  present,  recovered  without  tracheotomy.  ^^^^^  to  gen- 
More  visible  was  the  effect  in  regard  to  the  general  ^""^^  symptoms. 
condition  of  the  patients,  characterized  by  pulse  and 
temperature.  In  fresh  cases  it  was  observed  that  both 
showed  a  critical  decrease  after  large  doses  of  serum. 
The  conclusion  regarding  the  efficacy  of  antitoxin, 
judged  by  the  condition  of  pulse  and  temperature  alone, 
is  however  sometimes  incorrect  ;  first,  because  a  high 
temperature  frequently  remains,  after  the  child  has 
entered  the  convalescent  stage,  and  second,  because  the 
fever  may  be  due  to  a  complication,  and  not  to  the  toxin 
of  diphtheria.  Pulse  and  temperature  should  therefore 
not  be  expected  to  decrease  rapidly,  even  if  the  antitox- 
ine  has  its  full  specific  effect.  A  better  sign  by 
which  to  judge  the  latter,  is  the  subjective  condition  of 
the  patient.  It  is  remarkable  how  in  cases  of  a  most 
severe  form,  evident  signs  of  distress  vanished  within 
twenty-four  hours  and  the  children  whose  physical  and 
mental  condition  was  previously  very  low,  showed 
strength  and  good  humor.  In  such  cases  it  was  natu- 
rally surprising  to  the  physician  in  charge  "  to  see 
children,  who  were  in  doubtful  condition  when  admitted, 


administration. 


62  BLOOD    SERUM  THERAPY  AND  ANTITOXINS. 

playing  with  their  toys  or  eating  with  apparently  good 
appetite  the  following  day."  Regarding  those  cases  which 
resulted  fatally,  notwithstanding  the  application  of  anti- 
toxin, it  seems  that  at  least  in  some  of  them,  the  dose 
was  not  adequate  to  the  gravity  existing,  and  we  may 
therefore  confidently  expect  still  better  results  than  so 
far  obtained,  after  experience  has  enabled  us  to  state 
with  more  accuracy  the  quantity  required  in  the  differ- 
ent forms  which  may  come  before  us. 
Rules  for  The    principal    conclusions    to  which    the  mentioned 

results  have  led  in  serum  therapy  are  : 

1.  The  chance  to  save  a  patient  is  the  better  the 
sooner  antitoxin  is  applied. 

2.  The  seriousness  of  a  diphtheria  infection  is  fre- 
quently underestimated,  and  for  this  reason  it  seems  ad- 
visable to  apply  rather  large  than  insufficient  doses,  for 
light  cases  200  to  300  immunizing  units,  for  severe  ones, 
especially  after  tracheotomy,  500  to  800  units. 

3.  The  application  of  the  serum  has  to  be  repeated, 
according  to  the  gravity  of  the  case,  on  the  next  day  or 
even  on  the  same  day  until  a  quantity  of  600  to  1,000  or 
more  units  has  been  injected. 

4.  The  administration  of  serum  of  high  potence  is 
preferable  to  the  corresponding  amount  of  a  weaker  prep- 
aration. 

According  to  this,  Behring,  whose  method  we  have 
adopted,  recommends  a  serum  of  sixty  immunizing  units, 
which  is  supplied  in  bottles  of  three  different  sizes,  each 
being  one  dose.  No.  1  representing  600  units  to  be  applied 
only  on  the  first  or  second  day  of  the  disease,  No.  2  con- 
taining 1,000  units  to  be  used  in  more  serious  cases  on  the 
first  or  second  day  or  in  less  severe  cases  of  longer  stand- 
ing. No.  3  possesses  1,500  to  1,600  units  and  is  meant  for 
adults  or  very  severe  cases  in  children. 


DIPHTHERIA.  63 

In  case  the  serum  is  not  intended  as  a  curative  but  as  ^"t^^px,^^  ^^ » 

prophylactic. 

a  prophylactic,  the  strength  and  quantity  required  is  con- 
siderably smaller.  In  the  average  100  to  200  immunizing 
units  are  sufficient  to  protect  a  healthy  child  against  the 
infection.  How  long  such  protection  lasts  has  not  been 
fully  determined,  and  Kossel  advises  a  reinoculation  after 
two  to  three  weeks  if  the  child  is  still  exposed  to  infection. 
Such  small  doses,  however,  are  insufficient  if  the  child  is 
in  the  period  of  incubation,  which  would  put  it  under  the 
class  of  an  early  stage.  It  sometimes  may  happen  that 
children  are  infected,  but  for  lack  of  visible  symptoms  do 
not  get  an  adequate  dose,  in  which  case  the  failure  is  to 
be  attributed  to  the  fault  of  judgment  and  not  to  the  ineffi- 
ciency of  the  serum.  The  question  regarding  the  dose 
required  for  prophylactic  purposes  has  been  discussed  dur- 
ing the  last  few  months  with  considerable  interest.  In  a 
recent  publication  Behring  shows  how  he  came  to  use  the 
quantity  now  recommended  for  immunization.  Starting 
with  one  to  five  immunizing  units  in  districts  where  the 
schools  had  to  be  closed  on  account  of  an  epidemy,  he  soon 
was  convinced  of  the  insufficiency  of  such  small  doses. 
Better  results  were  obtained  with  fifteen  units,  but  in  the 
stage  of  incubation  even  this  dose  proved  inadequate.  A 
further  increase  to  sixty  units  gave  uniformly  good  results, 
and  the  fact  that  of  10,000  persons  inoculated  with  this 
amount  a  few  took  sick,  would  not  justify  us  in  assuming 
that  a  larger  quantity  is  required  as  the  normal  dose  for 
immunizatign. 

But  another  point  has  to  be  considered  in  regard  to  Duration  of 
this  question.  As  the  immunity  gradually  decreases  ac- 
cording to  the  elimination  of  the  antitoxin,  if  no  additional 
injections  are  given,  it  is  evident  that  its  duration  also  de- 
pends upon  the  quantity  first  administered.  On  the  other 
hand,  one  can   maintain  an   immunity  for   a    longer  period 


64  BLOOD  SERUM  THERAPY  AND  ANTITOXINS. 

by  repeated  application  of  smaller  doses  in  certain  inter- 
vals. For  this  reason  a  dose  not  over  150  immunizing 
units  has  been  recommended  by  Behring  for  the  purpose 
of  immunizing  healthy  persons.  If  the  serum  in  this  quan- 
tity or  any  other,  which  future  experience  might  determine, 
comes  up  to  our  expectation  regarding  its  immunizing 
power,  this  method  will  prove  to  be  the  strongest  factor 
to  prevent  the  disease.  A  most  essential  auxiliary  will 
always  be  an  early  diagnosis  and  the  immediate  application 
of  an  appropriate  local  treatment. 
Production  of  Xo  assist    in    the    complete    understanding  of  serum 

antitoxin.  ^  ^  ^ 

therapy  a  few  more  words  may  be  said  about  the  present 
theories  regarding  the  production  of  antitoxin,  and  its 
effect  upon  the  toxin  in  the  organism.  What  has  been 
said  in  regard  thereto  in  the  chapter  of  tetanus  applies  as 
well  to  diphtheria.  Comparing  the  opinion  first  expressed 
by  Behring  and  Kitasato,  that  the  toxin  and  antitoxin 
have  a  direct  effect  upon  each  other  because  of  their  being 
neutralized  when  mixed  in  a  test  tube  in  certain  propor- 
tions, and  the  opinion  of  Buchner  and  others  that  the  effi- 
cacy of  antitoxin  must  be  attributed  to  an  action  of  the 
living  cells,  we  will  find  that  either  of  these  opinions  is 
correct  to  a  certain  extent.  The  cells  play  undoubtedly 
an  important  part  in  effecting  the  results  observed,  but  at 
the  same  time  a  direct  antagonistic  action  must  exist  be- 
tween toxin  and  antitoxin.  This  is  corroborated  by  the 
test  which  shows  how  an  inoculation  with  a  mixture  of 
toxin  and  antitoxin  compares  with  the  separate  application 
of  the  two  substances.  In  the  former  case  two  parts  of  the 
antitoxin  mixed  with  the  toxin  proved  sufficient  to  neutralize 
the  toxin,  while  five  times  as  much  was  necessary  to  save  the 
animal  if  both  substances  were  injected  on  different  places. 
Action  of  cells  The  most  evident    proof  for   the  action  of  the  cells  is 

don/''™'''''^^    the  effect  of  a  mixture  of  toxin  and  antitoxin  upon  different 


DiPHTIJEkrA.  65 

species  of  animals.  If  these  substances  were  neutralized 
in  vitreo,  the  toxins  could  have  no  effect  upon  any  animal 
injected  with  such  mixture.  It  has  been  found,  however, 
that  if  such  mixture  was  ineffective  for  instance  upon 
mice,  it  still  acted  as  a  poison  upon  guinea  pigs  on  account 
of  their  higher  susceptibility.  Therefore,  one  cannot  deny 
that  the  cells  are  instrumental  in  producing  the  antitoxin. 
If  the  production  of  the  antitoxin  would  belong  to  the 
blood,  the  serum  of  an  animal  could  not  very  well  main- 
tain any  immunizing  power  after  a  quantity  as  large  as  the 
animal's  capacity  had  been  drawn.  The  serum,  however, 
retains  not  onl}/  its  previous  immunizing  value,  but  shows 
an  increase  of  the  latter  after  every  additional  application 
of  poison.  The  latter,  therefore,  seems  to  act  as  a  stimu- 
lant to  the  antitoxin  producing  cells. 

The  idea  that  antitoxin  is  a  cell  product  is  further 
substantiated  b}^  F.  Klemperer's  observation  that  in  immu- 
nized chickens  the  yolk  but  not  the  white  of  the  egg  has 
antitoxic  properties. 

With  the  above  explanations  we  have  outlined  the 
principal  points  of  serum  therapy  as  far  as  they  are  of 
interest  to  the  practitioner,  and  in  conclusion  wish  to 
make  a  few  remarks  which  may  be  of  practical  value. 

With  reference  to  the  preparation  of  the  serum  it 
should  be  borne  in  mind  that  in  order  to  obtain  a  serum 
of  high  value,  the  animal  which  is  to  furnish  the  serum 
must  be  inoculated  with  either  highly  virulent  cultures  or 
a  strong  toxin.  If  the  former  is  used  it  has  to  be  fre- 
quently tested  for  the  degree  of  its  virulence,  which  is 
known  to  change  from  very  slight  causes.  We  have  ob 
served  that  a  temporary  placing  of  the  culture  in  a  re- 
frigerator decreases  its  virulence  to  a  considerable  extent, 
and  that  even  a  difference  in  the  temperature  of  the  lab- 
oratory sometimes  leads  to  very  different  results  concern- 
ing the  efficacy  of  a  culture. 


66  BLOOD  SERUM  rilKRAPY  AND  ANTITOXIN. 

Of  equal  importance  is  the  proper  composition  of  the 
nutrient  substrate,  and  its  reaction  which  even  after  be- 
ing properly  prepared  is  subject  to  alterations  caused 
by  the  development  of  the  germs.  All  these  difficulties 
are  avoided  if  ready  toxin  of  a  tested  strength  is  used 
for  the  inoculations  because  its  virulence  is  not  altered  by 
cold  or  other  atmospheric  influences.  Furthermore  the 
serum  of  the  animal  herewith  intoxicated  must  be  tested  as 
to  its  value  on  a  series  of  animals  after  each  injection,  and 
also  each  time  it  has  to  furnish  serum  for  therapeutic  pur- 
poses. This  is  the  most  important  part  in  the  preparation 
of  the  antoxin,  because  the  clinical  results  depend  upon  a 
correct  estimation  of  the  material  put  on  the  market. 

A  source  which  cannot  be  trusted  in  this  respect  should, 
therefore,  be  boycotted  by  the  profession.  The  result  of 
such  tests  should  be  stated  on  the  label  of  every  bottle  or 
vial  (cf.  sample),  giving  the  number  of  immunizing  units 
per  ccm.,  so  that  the  practitioner  is  able  to  tell  what  dose 
to  inject  without  depending  on  the  directions,  which  cannot 
apply  to  every  case  uniforml}^  It  seems  advisable  to  fur- 
nish at  least  two  different  sizes  of  the  material,  one  for 
prophylactic  and  one  for  curative  purposes,  each  represent- 
ing one  dose  in  order  to  avoid  a  possible  infection  of  the 
serum  by  repeated  opening  of  the  bottle.  With  reference 
to  the  effectual  suppression  of  diphtheria  epidemics,  the 
sanitary  officers  should  cooperate  with  the  practitioner  by 
publishing  and  distributing,  especially  among  the  laboring 
classes,  such  instructions  as  would  inform  them  of  the  pos- 
sibility to  prevent  the  disease,  and  to  call  their  attention  to 
the  necessary  therapeutic  measures.  At  the  same  time 
these  instructions  would  help  to  overcome  the  prejudice, 
so  common  among  the  lower  classes,  against  any  opera- 
tive procedure. 


DIPHTHERIA.  67 

We  advise  a  distribution  of  a  circular  with  the  follow- 
ing information  : 

1.  Diphtheria  is  an  infectious  disease  most  dangerous 
to  children,  which  can  be  cured  by  early  attendance, 

2.  The  usual  symptoms  are  first  a  swelling  of  the 
tonsils,  commonly  known  as  sore  throat.  Soon  after  the 
inflammation  of  the  tonsils  white  spots  appear  on  the  latter, 
causing  destruction  of  the  tissue  and  frequently  obstructing 
the  air  passages. 

3.  These  white  spots  or  membranes  contain  germs 
which,  when  expectorated,  may  infect  other  persons  with 
the  disease. 

4.  With  the  inflammation  of  the  tonsils  fever  and 
general  discomfort  set  in. 

5.  From  the  tonsils  the  disease  frequently  extends  to 
the  nose,  from  which  a  suppurative  fluid  is  discharged,  and 
to  the  throat  and  lungs,  causing  hoarseness  and  difficulty  in 
breathing. 

6.  The  further  effect  of  the  disease  is  inflammation  of 
the  lungs,  kidneys,  and  a  general  poisoning  of  the  system, 
which  may  result  in  a  sudden  paralysis  of  the  heart. 

A  cure  of  this  disease  can  be  effected  almost  with  cer- 
tainty when  the  treatment  is  applied  in  the  incipient  stage 
— that  is,  the  first,  second  or  third  day  after  the  earliest 
symptoms  appeared.  In  advanced  cases  at  least  a  diminu- 
tion of  the  gravity  and  the  danger  to  infect  others  may  be 
effected  by  the  same  treatment.  It  is,  therefore,  the  duty 
of  the  parents  or  guardians  of  children  suffering  of  diphthe- 
ria to  apply  at  once  after  .discovery  of  the  first  symptoms  to 
the  proper  authority  for  attendance. 

1.  The  treatment  consists  in  hypodermic  injections  of 
a  remedy  called  '^  antitoxin,"  which  has  the  power  to  nullify 
the  poisonous  effect  of  the  disease.  This  remedy,  as  well 
as  its  application,  are  perfectly  harmless. 


68  BLOOD  SERUM  THERAPY  AND  ANTITOXIN. 

2.  With  the  same  remedy  in  smaller  dose  other  mem- 
bers of  the  family  can  be  protected  against  the  disease. 

3.  The  sick  child  should,  if  possible,  be  isolated  from 
others. 

4.  All  clothes  and  bed  linen  used  in  the  sick  room 
must  not  be  moved  from  the  room  or  used  before  being 
sterilized   according  to  the  directions  of  a  physician. 

5.  The  person  who  attends  to  the  child  should  fre- 
quently wash  his  hands  and  face  with  a  disinfecting  solu- 
tion as  prescribed  by  the  attending  physician. 

6.  All  dishes  and  table  supplies  used  by  the  child 
should  be  placed  in  boiling  water  immediately  after  use. 

We  are  confident  that  such  measure  will  prove  of  val- 
uable assistance  in  securing  satisfactory  results  from  the  use 
of  antitoxin,  as  the  cooperation  of  the  laity  is  necessary  to 
obtain  as  good  effects  as  were  accomplished  in  foreign  hos- 
pitals, in  which  the  largest  percentage  of  recover}^  occurred 
in  those  children  who  were  inoculated  in  the  earliest  stage 
of  the  disease.  The  enlightenment  of  the  public  on  this 
topic  will  undoubtedly  assure  still  better  statistics  regard- 
ing the  success  of  blood  serum  therapy.. 

Bibliography, 

Archiv.  f.  Klin.  "Chirurgie    1888-1892. 

Archiv.  of  Pedriatrics  Bd.  X.,  1893. 

Annales  de  1'  instil.,  Pasteur,  1890-94. 

Behring,  Blutserum  Therapie,  Th.  1   and  '2. 

Behring  and  Knorr,  Infection  and  Disinfection.  " 

Behring,  gesammelte  Abhandlungen. 

Berliner  Klin.  Woch.,  1889-1894. 

Brieger,  Ptomaine  Bd.,  1,  2.  3. 

British  Med.  Journal,  1891-1894. 

Centralblatt  f.  Bacteriologie,  1887-1894. 

Deutsche  Med.  Woch.,  18S9-1894. 

Emmerich  and  Terboi,  Natur  der  Schutz  undHeilsubst  des  Blutes 

Frsenkel,  Bacterienkunde. 

Giornadell  R.  Accad.  d.  Med.  di.  Tarr.  1884. 

La  Riforma  med.,  1891-1894. 

Nicalaier  Inaug.  dissert.  Goettingen,  1885. 

Philadelphia  Med.  News,  1893. 

Zeitschr  f.  Hygiene  und  Infectionskr.,  1889-1891. 


DIPHTHEKIA.  69 

SAMPLES  OF   LABELS. 

NAME  OF  FIRM. 

DIPHTHERIA- ANTITOXIN 

CONTAINING 

30  IMMUNIZING  UNITS  PER  I  CCM. 


TO  BE  USED  HYPODERMICALLY  AS  ONE  DOSE  AS  A 
PROPHYLACTIC  AGAINST  DIPHTHERIA. 


NAME  OF  FIRM. 
lO     COM. 

DIPHTHERIA-ANTITOXIN 

CONTAINING 

50  IMMUNIZING  UNITS  PER  1  CCM. 


TO    BE    USED    HYPODERMICALLY    AS   ONE    DOSE    IN 
INCIPIENT  CASE  OF  DIPHTHERIA. 


NAME  OF  FIRM. 
20     COM. 


DIPHTHERIA- ANTITOXIN 

CONTAINING 

50  IMMUNIZING  UNITS  PER  1  CCM. 


TO    BE    USED    HYPODERMICALLY    AS   ONE    DOSE    IN 
ADVANCED  CASES  OF  DIPHTHERIA. 


